http://2007.igem.org/wiki/index.php?title=Special:Contributions/Francesca.bugane2&feed=atom&limit=50&target=Francesca.bugane2&year=&month=2007.igem.org - User contributions [en]2024-03-29T08:06:32ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/Bologna_University/hereBologna University/here2007-10-26T15:55:42Z<p>Francesca.bugane2: </p>
<hr />
<div>[[Image:Tabs1.jpg]]<br />
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[[Image:Tabs2.jpg]]<br />
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[[Image:Tabs4.jpg]]<br />
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[[Bologna#Mathematical Model | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Week_11Week 112007-10-26T15:53:07Z<p>Francesca.bugane2: </p>
<hr />
<div>::'''09/10/07'''<br />
*Miniprep for:<br />
<br />
-[http://partsregistry.org/Part:BBa_I763027 I763027];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763028 I763028];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763019 I763019];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_P0412 P0412];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_S03520 S03520];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_S0100 S0100];<br />
<br />
Digestion for:<br />
<br />
-[http://partsregistry.org/Part:BBa_I763027 I763027] with Xba/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763028 I763028] with Spe/Pst1 and with Xba/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763019 I763019] with Xba/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_P0412 P0412] with Eco/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_S03520 S03520] with Eco/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763021 I763021]+[http://partsregistry.org/Part:BBa_S0100 S0100] with Eco/Spe;<br />
*Band extraction from gel for all digestion;<br />
*We have problems with [http://partsregistry.org/Part:BBa_J52034 J52034] and with [http://partsregistry.org/Part:BBa_I763019 I763019].<br />
*Ligations for:<br />
-[http://partsregistry.org/Part:BBa_I763028 I763028] + [http://partsregistry.org/Part:BBa_I763007 I763007]<br />
<br />
-[http://partsregistry.org/Part:BBa_S0100 S0100] + [http://partsregistry.org/Part:BBa_J04431 J04431], because we want to understand if LacI operates well; <br />
<br />
-[http://partsregistry.org/Part:BBa_J06550 J06550] + [http://partsregistry.org/Part:BBa_J04631 J04631], with this ligation we want to understand if it operates well, because it doesn't leak.<br />
<br />
::'''09/11/07'''<br />
* Digestion for:<br />
-[http://partsregistry.org/Part:BBa_I763020 I763020] with Xba/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_J22101 J22101] with Xba/Pst1;<br />
<br />
<br />
<br />
::'''09/12/07'''<br />
* Control digestion before fluorescence tests. [[(photos)]]<br />
* Ligations for:<br />
-[http://partsregistry.org/Part:BBa_I763005 I763005] (Spe/Pst1) + [http://partsregistry.org/Part:BBa_J04031 J04031] (Xba/Pst1);<br />
<br />
-[http://partsregistry.org/Part:BBa_I763025 I763025] (Eco/Spe) + [http://partsregistry.org/Part:BBa_J04031 J04031] (Eco/Xba);<br />
<br />
-[http://partsregistry.org/Part:BBa_I763015 I763015] (Eco/Spe) + [http://partsregistry.org/Part:BBa_J04031 J04031] (Eco/Xba).<br />
<br />
<br />
::'''09/13/07'''<br />
<br />
*We have find any colonies for [http://partsregistry.org/Part:BBa_I763015 I763015] + [http://partsregistry.org/Part:BBa_J04031 J04031].<br />
*Miniprep for:<br />
-[http://partsregistry.org/Part:BBa_I763005 I763005] + [http://partsregistry.org/Part:BBa_J04031 J04031];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763025 I763025] + [http://partsregistry.org/Part:BBa_J04031 J04031];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763019 I763019].<br />
*Control digestion for [http://partsregistry.org/Part:BBa_I763019 I763019].<br />
*M9 medium and lactose stocks preparation.<br />
<br />
*'''Testing our devices'''<br />
<br />
We take 5ml of LB medium in which we put a colony of bacteria which contain the [http://partsregistry.org/Part:BBa_I763028 I763028] plasmid.<br />
We examine a drop of it with our fluorescence microscopy. In these conditions, the bacteria don’t beam fluorescence.<br />
Starting from this moment, we add into the LB medium 1mM of IPTG every 30 minutes until it reaches 4mM , checking each time for any possible fluorescence, with negative results. We then try another option: after diluting 1ml of the original solution with 4ml of LB, we add IPTG until this solution reaches 10mM, then 20mM and finally 40mM. Despite that, no fluorescence is visible.<br />
For this reason we then test single parts of the plasmid. We take three populations of [http://partsregistry.org/Part:BBa_I763019 I763019] bacteria and one of Plac-cI-GFP bacteria. We add 1mM of IPTG to every population to check for fluorescence. We see the three [http://partsregistry.org/Part:BBa_I763019 I763019] populations produce a photomultiplier output between 5.55 and 5.61Volts, while the Plac-cI-GFP population results more fluorescent giving a 5.91Volts output.<br />
Every time we check for fluorescence, we use, as said, not only the photo camera but also the photomultiplier. Every measurement shows a 0.18Volts offset due to environment light and a 1.30Volts offset due to the excitation light at 501nm. Since we know GFP gives a maximum of fluorescence when excited at 501nm, we decide to go on with this wavelength despite the offset (a change of excitation wavelength reduces both the offset and the signal).<br />
<br />
<br />
<br />
<br />
::'''09/14/07'''<br />
<br />
*Glicerol stocks for:<br />
-[http://partsregistry.org/Part:BBa_I763019 I763019];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763031 I763031];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763020 I763020];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763026 I763026].<br />
*Fluorescence test for:<br />
-[http://partsregistry.org/Part:BBa_I763019 I763019];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763031 I763031].<br />
<br />
*Miniprep for:<br />
-[http://partsregistry.org/Part:BBa_I763019 I763019];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763031 I763031];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763026 I763026];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763020 I763020].<br />
*Digestion for:<br />
-[http://partsregistry.org/Part:BBa_I763019 I763019] with Eco/Xba;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763026 I763026] with Eco/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763026 I763026] with Spe/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763026 I763026] with Eco/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763020 I763020] with Xba/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763031 I763031] with Xba/Pst1.<br />
*Band extraction from gel for all digestion except for [http://partsregistry.org/Part:BBa_I763031 I763031]. [[(photos2)]]<br />
<br />
<br />
<br />
<br />
[[Bologna#Diary | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Week_12Week 122007-10-26T15:52:40Z<p>Francesca.bugane2: </p>
<hr />
<div>::'''09/17/07'''<br />
*Ligations for:<br />
-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763019 I763019];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763004 I763004];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763025 I763025] + [http://partsregistry.org/Part:BBa_J04031 J04031].<br />
*We transform ligations and strake them on plates.<br />
<br />
<br />
<br />
::'''09/18/07'''<br />
*We inoculate a colony for yesterday ligations in 5ml of LB medium O/N.<br />
<br />
<br />
::'''09/19/07'''<br />
*Miniprep for:<br />
-[http://partsregistry.org/Part:BBa_I763028 I763028];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763027 I763027];<br />
<br />
-[http://partsregistry.org/Part:BBa_I763035 I763035] (Spe/Pst1), (Xba/Pst1).<br />
*Digestion for:<br />
-[http://partsregistry.org/Part:BBa_I763028 I763028] with Eco/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763027 I763027] with Eco/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763035 I763035] with Eco/Spe;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763007 I763007] with Eco/Xba;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763036 I763036] with Eco/Spe1;<br />
<br />
*Band extraction from gel for all digestion and then we observe:<br />
-[http://partsregistry.org/Part:BBa_I763028 I763028], [http://partsregistry.org/Part:BBa_I763027 I763027] are died;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763035 I763035] is correct;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763007 I763007] not found;<br />
<br />
-[http://partsregistry.org/Part:BBa_I763036 I763036] is correct.<br />
<br />
<br />
::'''09/20/07'''<br />
<br />
'''Testing our devices''' <br />
<br />
We wanted to test if our newly-constructed devices work, that is if they beam fluorescence when inducted with IPTG. To do so we performed 3 tests in parallel in 3 different tubes, that we name here A, B and C.<br />
::1. In tube A e in tube B we add 5ml of LB medium and 2 different colonies of [http://partsregistry.org/Part:BBa_I763019 I763019] plasmid. <br />
::2. In tube C we add 5ml of LB medium and a colony of [http://partsregistry.org/Part:BBa_I763031 I763031] plasmid. <br />
::3. After 2 hours we measure the OD value of tubes A, B, C and it is around 0.5. <br />
::4. For tubes A, B, C we divided the bacteria fluid in two different tubes A1, A2; B1, B2; C1, C2. <br />
::5. We add 1mM IPTG to the solution into tubes A2, B2, C2. <br />
* The analyzed fluid without IPTG (tubes A1, B1, C1) includes: <br />
::-2.5ml of original tube fluid; <br />
::-2.5ml of LB medium; <br />
::-2.5ul of kanamicin. <br />
*The analyzed fluid with IPTG (tube A2, B2, C2) includes: <br />
::-2.5ml of original tube fluid; <br />
::-2.5ml of LB medium; <br />
::-2.5ul of kanamicin; <br />
::-50ul of 100mM IPTG. <br />
::6. We examine a bacteria fluid drop of tubes A1, A2, B1, B2, C1, C2 with our fluorescence microscope. <br />
::7. The bacteria with [http://partsregistry.org/Part:BBa_I763019 I763019] plasmid doesn’t beam fluorescence with (A2, B2) nor without (A1, B1) IPTG; <br />
::8. Very few bacteria with [http://partsregistry.org/Part:BBa_I7630231 I763031] plasmid with (C2) and without (C1) IPTG beam fluorescence. <br />
*In conclusion, we see that bacteria with [http://partsregistry.org/Part:BBa_I763019 I763019] plasmid (tubes A1, B1, A2, B2) don't seem to work at all, and bacteria with [http://partsregistry.org/Part:BBa_I763031 I763031] plasmid (tube C1, C2) don't seem to work properly either. We plan other experiments for next week.<br />
<br />
<br />
<br />
<br />
::'''09/21/07'''<br />
Meeting: definition of fluorescence test protocol. <br />
<br />
<br />
<br />
[[Bologna#Diary | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Week_13Week 132007-10-26T15:51:56Z<p>Francesca.bugane2: </p>
<hr />
<div>::'''09/24/07'''<br />
*Ligation for [http://partsregistry.org/Part:BBa_I763031 I763031].<br />
*Transformation of [http://partsregistry.org/Part:BBa_I763031 I763031].<br />
*We inoculate [http://partsregistry.org/Part:BBa_I763036 I763036], [http://partsregistry.org/Part:BBa_I763007 I763007], [http://partsregistry.org/Part:pSB4A3 pSB4A3] in 5ml of LB.<br />
*Transformation of [http://partsregistry.org/Part:BBa_I763028 I763028].<br />
<br />
<br />
<br />
::'''09/25/07'''<br />
*Miniprep of:<br />
::- [http://partsregistry.org/Part:BBa_I763036 I763036];<br />
::- [http://partsregistry.org/Part:BBa_I763007 I763007];<br />
::- [http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
::- [http://partsregistry.org/Part:BBa_I763033 I763033]. <br />
*We inoculate (5ml LB, 5ul amp 1000X) straked transformations of:<br />
::- [http://partsregistry.org/Part:BBa_I763033 I763033]; <br />
::- [http://partsregistry.org/Part:BBa_I763028 I763028] (vitality test). <br />
<br />
*Transformations of:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031]. <br />
<br />
<br />
<br />
<br />
::'''09/26/07'''<br />
'''Fluorescence test according to [[Bologna University/Fluorescence Test1 | Fluorescence Test Protocol]]''' of:<br />
<br />
::1. ''[http://partsregistry.org/Part:BBa_I763004 I763004]'':<br />
::::-bacteria use to have the PMT output maximum value;<br />
::::-normalization of OD value at 0.5;<br />
::::-output value without IPTG: [0.22, 0.27]V;<br />
::::-output value with IPTG: <br />
::::::::-after 10min: [0.18,0.28]V;<br />
::::::::-after 20min: [0.41,0.46]V;<br />
::::::::-after 30min: [0.42,0.70]V;<br />
::::::::-after 60min: [0.48,0.80]V;<br />
::::-thus we decide to grow the OD value to have higher output value.<br />
<br />
::2. ''[http://partsregistry.org/Part:BBa_I763028 I763028]''<br />
::::- we observe bacteria vitality, but there isn’t fluorescence after O/N growth with and without progressive induction with IPTG (1mM, 2mM, 3mM, 10mM every 60min).<br />
::::- thus we decide to use lactose.<br />
<br />
<br />
<br />
<br />
::'''09/27/07'''<br />
'''Fluorescence test according to [[Bologna University/Fluorescence Test1 | Fluorescence Test Protocol]]''' of:<br />
::1. ''[http://partsregistry.org/Part:BBa_I763004 I763004]'':<br />
::::-output value like to value with OD=0.5.<br />
::2. ''[http://partsregistry.org/Part:BBa_I763028 I763028]'':<br />
::::-growth in LB medium;<br />
::::-pellet in M9 with lactose;<br />
::::-we observe them every 30min:<br />
::::::::-bacteria vitality confirmed;<br />
::::::::-bacteria don’t beam fluorescent. <br />
::3. ''[http://partsregistry.org/Part:BBa_I763031 I763031]'':<br />
::::-Output value without IPTG:0.05V;<br />
::::-Output value with IPTG: about 0V.<br />
::4. ''[http://partsregistry.org/Part:BBa_I763019 I763019]'':<br />
::::-Output value without IPTG:0.05V;<br />
::::-Output value with IPTG: about 0V.<br />
'''Conclusions:'''<br />
::-using of low copy plasmid;<br />
::-higher OD;<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032] and [http://partsregistry.org/Part:BBa_I763034 I763034] fluorescent test.<br />
*We inoculate [http://partsregistry.org/Part:BBa_I763033 I763033] and [http://partsregistry.org/Part:BBa_I763028 I763028].<br />
<br />
<br />
<br />
::'''09/28/07'''<br />
*Digestion of : [http://partsregistry.org/Part:BBa_I763033 I763033] with Eco/Spe.<br />
*Ligation with [http://partsregistry.org/Part:BBa_J04431 J04431] (Eco/Xba). <br />
*We transform and strake on plates.<br />
<br />
[[Bologna#Diary | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Week_14Week 142007-10-26T15:51:29Z<p>Francesca.bugane2: </p>
<hr />
<div>::'''10/01/07'''<br />
*Meeting: Ethical issue.<br />
*Work on image segmentation algorithm.<br />
<br />
<br />
::'''10/02/07'''<br />
*Digestion of [http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
*Run on electrophoresis gel.<br />
*[http://partsregistry.org/Part:pSB4A3 pSB4A3] and [http://partsregistry.org/Part:BBa_I763033 I763033] band extraction.<br />
*Ligations for: <br />
::-[http://partsregistry.org/Part:BBa_I763004 I763004]+[http://partsregistry.org/Part:BBa_I763033 I763033];<br />
::-[http://partsregistry.org/Part:BBa_I763012 I763012]+[http://partsregistry.org/Part:BBa_I763020 I763020][http://partsregistry.org/Part:BBa_I763032 (I763032)].<br />
<br />
<br />
<br />
<br />
::'''10/03/07'''<br />
*Digestions of:<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032] with Eco/Pst1;<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] with Eco/Pst1;<br />
::-[http://partsregistry.org/Part:BBa_I763036 I763036] with Eco/Pst1;<br />
::-[http://partsregistry.org/Part:pSB4A3 pSB4A3] with Eco/Pst1.<br />
*Run on electrophoresis gel.<br />
*[http://partsregistry.org/Part:BBa_I763031 I763031], [http://partsregistry.org/Part:BBa_I763036 I763036] and [http://partsregistry.org/Part:pSB4A3 pSB4A3] band extraction.<br />
*Transformations of:<br />
::-[http://partsregistry.org/Part:BBa_I763034 I763034]; <br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031];<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032].<br />
*Ligations for:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031]+[http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032]+[http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
<br />
'''Experimental Setup'''<br />
<br />
*To have an higher precision for our measurements, we have decided to get, for the same field of view on the same sample of bacteria solution, either a short film segment (from which we can extract frames) and a Microsoft Excel file, which contains a ten seconds interval of values from the photomultiplier output, taken at a 100Hz frequency.<br />
<br />
*In order to have the same focus for the microscope's oculars, the photocamera and the photomultiplier we have to calibrate our system by changing the position of both camera and photomultiplier.<br />
<br />
*Finally, we check that the photomultiplier field of view is the same size as the microscope's one: by moving the appropiate diaphragms, we adjust the previously done settings. <br />
<br />
<br />
<br />
::'''10/04/07'''<br />
*Trasformation of:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031]+[http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032]+[http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
*We inoculate [http://partsregistry.org/Part:BBa_I763034 I763034], [http://partsregistry.org/Part:BBa_I763031 I763031] + [http://partsregistry.org/Part:pSB4A3 pSB4A3] and [http://partsregistry.org/Part:BBa_I763032 I763032]+[http://partsregistry.org/Part:pSB4A3 pSB4A3] (in 6 ml).<br />
<br />
<br />
<br />
<br />
::'''10/05/07'''<br />
*Stock preparation (500 ul) of [http://partsregistry.org/Part:I763034 I763034],[http://partsregistry.org/Part:BBa_I763031 I763031]+[http://partsregistry.org/Part:BBa_pSB4A3 pSB4A3] and [http://partsregistry.org/Part:BBa_I763032 I763032]+[http://partsregistry.org/Part:BBa_pSB4A3 pSB4A3].<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763034 I763034], [http://partsregistry.org/Part:BBa_I763031 I763031]+[http://partsregistry.org/Part:BBa_pSB4A3 pSB4A3] and [http://partsregistry.org/Part:BBa_I763032 I763032]+[http://partsregistry.org/Part:BBa_pSB4A3 pSB4A3].<br />
*Control digestion of: <br />
::-[http://partsregistry.org/Part:BBa_I763034 I763034] with Xba/Pst1;<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] with Eco/Xba and Xba/Pst1;<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032] with Eco/Xba and Xba/Pst1.<br />
*Run on electrophoresis gel and [http://partsregistry.org/Part:BBa_I763032 I763032] (Eco/Xba and Xba/Pst1) band extraction.<br />
<br />
<br />
<br />
[[Bologna#Diary | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Week_15Week 152007-10-26T15:51:10Z<p>Francesca.bugane2: </p>
<hr />
<div>::'''10/08/07'''<br />
*We inoculate:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid);<br />
::-E.Coli;<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032] (high copy plasmid).<br />
*Digestion of:<br />
::-[http://partsregistry.org/Part:BBa_I763035 I763035] with Eco/Pst1;<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007] with Eco/spe;<br />
::-[http://partsregistry.org/Part:BBa_I763026 I763026] with Eco/Xba;<br />
::-[http://partsregistry.org/Part:pSB4A3 pSB4A3] with Spe/Xba.<br />
*Ligation for:<br />
::-[http://partsregistry.org/Part:BBa_I763035 I763035]+[http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007]+[http://partsregistry.org/Part:BBa_I763026 I763026];<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007]+[http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032]+[http://partsregistry.org/Part:pSB4A3 pSB4A3].<br />
*Run on electrophoresis gel.<br />
*[http://partsregistry.org/Part:BBa_I763007 I763007] and [http://partsregistry.org/Part:BBa_I763026 I763026] band extraction.<br />
*O/N ligation for:<br />
::-[http://partsregistry.org/Part:BBa_I763026 I763026]+[http://partsregistry.org/Part:BBa_I763007 I763007];<br />
::-[http://partsregistry.org/Part:BBa_I763024 I763024]+[http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032]+[http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007]+[http://partsregistry.org/Part:pSB4A3 pSB4A3].<br />
*We inoculate:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031](low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016](low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032] (high copy plasmid).<br />
<br />
<br />
<br />
::'''10/09/07'''<br />
*Transformation of:<br />
::-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763007 I763007];<br />
::-[http://partsregistry.org/Part:BBa_I763024 I763024] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007] (low copy plasmid).<br />
*We inoculate in 6ml:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid).<br />
*Ligation for:<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (Eco/Spe)+GFP.(Eco/Xba).<br />
<br />
<br />
::'''10/10/07'''<br />
*Miniprep of:<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] (low copy plasmid).<br />
*Digestion for :<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] with Xba/Pst1;<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid) with Spe/Pst1;<br />
*Run on electrophoresis gel and [http://partsregistry.org/Part:I763016 I763016] band extraction.<br />
*Ligation for :<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid)+GFP;<br />
::-[http://partsregistry.org/Part:BBa_I763024 I763024]+[http://partsregistry.org/Part:pSB4A3 pSB4A3];<br />
::-[http://partsregistry.org/Part:BBa_I763024 I763024]+[http://partsregistry.org/Part:BBa_C0012 C0012] (high copy plasmid).<br />
*We inoculate in 6ml:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763007 I763007](high copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763024 I763024] (low copy plasmid).<br />
::'''10/11/07'''<br />
*Stock preparation (500ul) of:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763007 I763007](high copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763024 I763024] (low copy plasmid).<br />
*Miniprep of:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763026 I763026]+ [http://partsregistry.org/Part:BBa_I763007 I763007](high copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763024 I763024] (low copy plasmid).<br />
*Fluorescence test of:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763007 I763007] (high copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007] (low copy plasmid).<br />
*Control digestion of:<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] with Xba/Pst1;<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] with Xba/Pst1 and Spe/Xba;<br />
::-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763007 I763007] with Xba/Pst1 and Spe/Xba;<br />
::-[http://partsregistry.org/Part:BBa_I763024 I763024] with Xba/Pst1;<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007] with Xba/Pst1.<br />
*Run on electrophoresis gel and band extraction.<br />
*Transformation of:<br />
::-[http://partsregistry.org/Part:BBa_I763019 I763019];<br />
::-[http://partsregistry.org/Part:BBa_I763024 I763024]+[http://partsregistry.org/Part:BBa_C0012 C0012];<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032].<br />
*We inoculate <br />
::-[http://partsregistry.org/Part:BBa_I763019 I763019]. <br />
::'''10/12/07'''<br />
*Miniprep of:<br />
::-[http://partsregistry.org/Part:BBa_I763019 I763019].<br />
*Digestion of:<br />
::-[http://partsregistry.org/Part:BBa_I763019 I763019] with Xba/Pst1;<br />
::-[http://partsregistry.org/Part:BBa_I763019 I763019] with Eco/Xba.<br />
*Run on electrophoresis gel.<br />
*[http://partsregistry.org/Part:BBa_I763019 I763019] (Xba/Pst1) and [http://partsregistry.org/Part:BBa_I763019 I763019] (Eco/Xba) band extraction.<br />
<br />
<br />
<br />
[[Bologna#Diary | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Week_16Week 162007-10-26T15:50:49Z<p>Francesca.bugane2: </p>
<hr />
<div>::'''10/15/07'''<br />
*O/N ligations for:<br />
::-[http://partsregistry.org/Part:BBa_I763019 I763019];<br />
::-[http://partsregistry.org/Part:BBa_I763039 I763039].<br />
<br />
<br />
<br />
<br />
::'''10/16/07'''<br />
*Transformation of [http://partsregistry.org/Part:BBa_I763019 I763019] and [http://partsregistry.org/Part:BBa_I763039 I763039].<br />
*We inoculate:<br />
::-[http://partsregistry.org/Part:BBa_I763005 I763005];<br />
::-[http://partsregistry.org/Part:BBa_I763015 I763015];<br />
::-[http://partsregistry.org/Part:BBa_I763004 I763004].<br />
*Trasformation of [http://partsregistry.org/Part:BBa_I763019 I763019], subsequently growth in 2 mL of LB medium to test if fresh transformed cells have the same problem of vitality seen using the stock.<br />
<br />
<br />
::'''10/17/07'''<br />
*Miniprep of:<br />
::-[http://partsregistry.org/Part:BBa_I763005 I763005];<br />
::-[http://partsregistry.org/Part:BBa_I763015 I763015];<br />
::-[http://partsregistry.org/Part:BBa_I763004 I763004].<br />
*We inoculate:<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029];<br />
::-[http://partsregistry.org/Part:BBa_I763039 I763039] (high copy).<br />
*Control Digestion of the following Minipreps for results impagination:<br />
::-[http://partsregistry.org/Part:BBa_I763020 I763020];<br />
::-[http://partsregistry.org/Part:BBa_I763004 I763004];<br />
::-[http://partsregistry.org/Part:BBa_I763011 I763011];;<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007] (on high copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763007 I763007] (on low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763033 I763033];<br />
::-[http://partsregistry.org/Part:BBa_I763015 I763015];<br />
::-[http://partsregistry.org/Part:BBa_I763012 I763012];<br />
::-[http://partsregistry.org/Part:BBa_I763005 I763005];<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031].<br />
*Gel electrophoresis of the fragments above.<br />
<br />
<br />
<br />
::'''10/18/07'''<br />
*Miniprep:<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029]; <br />
::-[http://partsregistry.org/Part:BBa_I763039 I763039] (high copy). <br />
<br />
*Digestion for:<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] (Eco/Pst1)<br />
<br />
*Band extraction of:<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] (Eco/Pst1)<br />
<br />
*Ligation for:<br />
::-[http://partsregistry.org/Part:pSB4A3 pSB4A3] (Eco/Pst1)<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] (Eco/Pst1)<br />
<br />
*Ligation for:<br />
::-[http://partsregistry.org/Part:BBa_I763004 I763004] (Xba/Pst1)<br />
::-[http://partsregistry.org/Part:BBa_I763026 I763026] (Spe/Pst1)<br />
<br />
*Control Digestion of the following Minipreps for results impagination:<br />
::- [http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid);<br />
::- [http://partsregistry.org/Part:BBa_I763016 I763016] (high copy plasmid);<br />
::- [http://partsregistry.org/Part:BBa_I763036 I763036];<br />
::- [http://partsregistry.org/Part:BBa_I763019 I763019];<br />
::- [http://partsregistry.org/Part:BBa_I763032 I763032];<br />
::- [http://partsregistry.org/Part:BBa_I763025 I763025] (low copy plasmid);<br />
::- [http://partsregistry.org/Part:BBa_I763025 I763025] (high copy plasmid);<br />
::- [http://partsregistry.org/Part:BBa_I763026 I763026];<br />
::- [http://partsregistry.org/Part:BBa_I763035 I763035]. <br />
<br />
*Gel electrophoresis of the fragments above.<br />
<br />
<br />
<br />
::'''10/19/07'''<br />
*Transformation:<br />
::-[http://partsregistry.org/Part:BBa_I763034 I763034] (on high copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763039 I763039];<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] on [http://partsregistry.org/Part:pSB4A3 pSB4A3] (low copy plasmid).<br />
<br />
<br />
<br />
<br />
[[Bologna#Diary | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Week_17Week 172007-10-26T15:50:27Z<p>Francesca.bugane2: </p>
<hr />
<div>::'''10/22/07'''<br />
*Ligation:<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032] (on [http://partsregistry.org/Part:pSB4A3 pSB4A3]);<br />
<br />
*Transformation:<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032] (on [http://partsregistry.org/Part:pSB4A3 pSB4A3]);<br />
<br />
*We inoculate:<br />
::-[http://partsregistry.org/Part:BBa_I763034 I763034] (on high copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763031 I763031] (on low copy plasmid), 3 different inocules using 3 different colonies;<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] on [http://partsregistry.org/Part:pSB4A3 pSB4A3] (high copy plasmid).<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] on [http://partsregistry.org/Part:pSB4A3 pSB4A3] (low copy plasmid).<br />
<br />
<br />
<br />
<br />
::'''10/23/07'''<br />
*Miniprep:<br />
::-[http://partsregistry.org/Part:BBa_I763034 I763034] (on high copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] (high copy plasmid).<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] (low copy plasmid).<br />
<br />
*Control Digestion for results impagination:<br />
::-[http://partsregistry.org/Part:BBa_I763016 I763016] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763036 I763036];<br />
::-[http://partsregistry.org/Part:BBa_I763019 I763019];<br />
::-[http://partsregistry.org/Part:BBa_I763032 I763032];<br />
::-[http://partsregistry.org/Part:BBa_I763025 I763025] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763027 I763027];<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] (low copy plasmid);<br />
::-[http://partsregistry.org/Part:BBa_I763029 I763029] (high copy plasmid).<br />
<br />
*Inoculate [http://partsregistry.org/Part:BBa_I763032 I763032] (on [http://partsregistry.org/Part:pSB4A3 pSB4A3]).<br />
<br />
<br />
<br />
<br />
::'''10/24/07'''<br />
<br />
Data elaboration and wiki updating<br />
<br />
<br />
::'''10/25/07'''<br />
<br />
Data elaboration and wiki updating<br />
<br />
<br />
::'''10/26/07'''<br />
<br />
Data elaboration and wiki updating<br />
<br />
<br />
[[Bologna#Diary | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:47:50Z<p>Francesca.bugane2: /* Introduction */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
<br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath_def.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
<br />
[[Image:batteri1.jpg|center]]<br />
<br />
Figure 11.''Fluorescent bacteria''<br />
<br />
<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:batteri2.jpg|center]]<br />
<br />
<br />
Figure 12.''Algorithm application.''<br />
<br />
<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:47:09Z<p>Francesca.bugane2: </p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath_def.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
<br />
[[Image:batteri1.jpg|center]]<br />
<br />
Figure 11.''Fluorescent bacteria''<br />
<br />
<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:batteri2.jpg|center]]<br />
<br />
<br />
Figure 12.''Algorithm application.''<br />
<br />
<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:45:26Z<p>Francesca.bugane2: /* Image Elaboration */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath_def.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
<br />
[[Image:batteri1.jpg|center]]<br />
<br />
Figure 11.''Fluorescent bacteria''<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:batteri2.jpg|center]]<br />
<br />
<br />
Figure 12.''Algorithm application.''<br />
<br />
<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:45:12Z<p>Francesca.bugane2: /* Image Elaboration */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
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"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
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"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
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<br />
''(Aforisma di Ippocrate di Coo)''<br />
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<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
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<br />
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<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
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So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
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[[Image:switch_2.jpg|center]]<br />
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Figure2.''The "smart" switch.''<br />
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This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
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<br />
[[Image:scaldabagno3.jpg|center]]<br />
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Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
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The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
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[[Image:schema1.jpg|center]]<br />
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Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
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As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
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'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
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'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
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'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
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[[Image:introduz.jpg|center]]<br />
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Figure 5. ''Schematic view of our device''<br />
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==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
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[[Image:modellomath_def.jpg|center]]<br />
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Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
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<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
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[[Image:StateIng.jpg|center]]<br />
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No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
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<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
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Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
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All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
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[[Image:Iptg_Gfp3.jpg|center]]<br />
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Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
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[[Image:Stability_plane2.jpg|center]]<br />
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Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
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[[Image:grafico3.jpg|center]]<br />
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Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
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==Image Acquisition and Elaboration==<br />
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===Experimental SetUp===<br />
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[[Image:Microscopy.jpg|center]]<br />
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Figure 10. Image acquisition system based on the fluorescence microscope.<br />
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The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
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The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
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The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
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The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
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The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
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===Image Acquisition===<br />
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Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
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[[Image:batteri1.jpg|center]]<br />
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Figure 11.''Fluorescent bacteria''<br />
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===Image Elaboration===<br />
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Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
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Here are the processing phases:<br />
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*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
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[[Image:batteri2.jpg|center]]<br />
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Figure 12.''Algorithm application.''<br />
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*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
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This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
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===Components===<br />
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The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
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[[Image:Tutto3.jpg]]<br />
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''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
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:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
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:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
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:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
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[[Image:no_IPTG.jpg]]<br />
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''Figure 14: Project in action: no IPTG in the medium.''<br />
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For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
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[[Image:con_IPTG1.jpg]]<br />
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''Figure 15: Project in action: IPTG in the medium.''<br />
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===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
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-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Batteri2.jpgFile:Batteri2.jpg2007-10-26T15:43:22Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:43:10Z<p>Francesca.bugane2: /* Image Elaboration */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath_def.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
<br />
[[Image:batteri1.jpg|center]]<br />
<br />
Figure 11.''Fluorescent bacteria''<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:batteri2.jpg|center]]<br />
<br />
<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:42:22Z<p>Francesca.bugane2: /* Image Elaboration */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath_def.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
<br />
[[Image:batteri1.jpg|center]]<br />
<br />
Figure 11.''Fluorescent bacteria''<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:batteri2|center]]<br />
<br />
<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/FP | Francesco Pasqualini]] ::[[Bologna_University/GC | Guido Costa]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:40:43Z<p>Francesca.bugane2: /* Image Acquisition */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
<br />
[[Image:batteri1.jpg|center]]<br />
<br />
Figure 11.''Fluorescent bacteria''<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/FP | Francesco Pasqualini]] ::[[Bologna_University/GC | Guido Costa]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Batteri1.jpgFile:Batteri1.jpg2007-10-26T15:40:05Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:39:34Z<p>Francesca.bugane2: /* Image Acquisition */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
<br />
[[Image:batteri1.jpg|center]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] <br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:31:36Z<p>Francesca.bugane2: /* Image Elaboration */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] <br />
:'''Graduate Student'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:28:43Z<p>Francesca.bugane2: /* Experimental SetUp */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] <br />
:'''Graduate Student'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:24:17Z<p>Francesca.bugane2: /* About Us */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
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<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
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"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
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<br />
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<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
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<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
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Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
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Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] <br />
:'''Graduate Student'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Grafico3.jpgFile:Grafico3.jpg2007-10-26T15:07:17Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:06:55Z<p>Francesca.bugane2: /* Simulation */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], graduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], graduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] <br />
:'''Graduate Student'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Stability_plane2.jpgFile:Stability plane2.jpg2007-10-26T15:06:18Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:05:40Z<p>Francesca.bugane2: /* Simulation */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], graduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], graduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane2.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] <br />
:'''Graduate Student'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Iptg_Gfp3.jpgFile:Iptg Gfp3.jpg2007-10-26T15:04:53Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:04:05Z<p>Francesca.bugane2: /* Simulation */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], graduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], graduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] <br />
:'''Graduate Student'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T15:00:57Z<p>Francesca.bugane2: /* Overview Table */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], graduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], graduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp2.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] <br />
:'''Graduate Student'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T14:56:28Z<p>Francesca.bugane2: /* Model */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp2.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/IB| Iros Barozzi]] <br />
:'''Graduate Student''' <br />
::[[Bologna_University/AP | Alice Pasini]] <br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
:: Sara Montagna • [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T14:56:06Z<p>Francesca.bugane2: /* Model */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
No linear function: <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp2.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/IB| Iros Barozzi]] <br />
:'''Graduate Student''' <br />
::[[Bologna_University/AP | Alice Pasini]] <br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
:: Sara Montagna • [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T14:55:43Z<p>Francesca.bugane2: /* Model */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
'''No linear function''': <br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp2.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/IB| Iros Barozzi]] <br />
:'''Graduate Student''' <br />
::[[Bologna_University/AP | Alice Pasini]] <br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
:: Sara Montagna • [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Equaz8.jpgFile:Equaz8.jpg2007-10-26T14:53:40Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Bologna_University/Plot4Bologna University/Plot42007-10-26T14:53:29Z<p>Francesca.bugane2: </p>
<hr />
<div>[[Image:Equaz8.jpg|center]]<br />
<br />
[[Bologna#Model | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Bologna_University/Plot3Bologna University/Plot32007-10-26T14:53:03Z<p>Francesca.bugane2: </p>
<hr />
<div>[[Image:Equaz7.jpg|center]]<br />
<br />
[[Bologna#Model | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Equaz7.jpgFile:Equaz7.jpg2007-10-26T14:52:46Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Bologna_University/Plot3Bologna University/Plot32007-10-26T14:52:36Z<p>Francesca.bugane2: </p>
<hr />
<div>[[Image:Equaz7.jpg]]<br />
<br />
[[Bologna#Model | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Bologna_University/Plot2Bologna University/Plot22007-10-26T14:52:14Z<p>Francesca.bugane2: </p>
<hr />
<div>[[Image:Equaz6.jpg|center]]<br />
<br />
[[Bologna#Model | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Equaz6.jpgFile:Equaz6.jpg2007-10-26T14:51:55Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Bologna_University/Plot2Bologna University/Plot22007-10-26T14:51:45Z<p>Francesca.bugane2: </p>
<hr />
<div>[[Image:Equaz6.jpg]]<br />
<br />
[[Bologna#Model | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Bologna_University/Plot1Bologna University/Plot12007-10-26T14:51:15Z<p>Francesca.bugane2: </p>
<hr />
<div>[[Image:Equaz5.jpg|center]]<br />
<br />
<br />
[[Bologna#Model | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Equaz5.jpgFile:Equaz5.jpg2007-10-26T14:50:54Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/Bologna_University/Plot1Bologna University/Plot12007-10-26T14:50:33Z<p>Francesca.bugane2: </p>
<hr />
<div>[[Image:Equaz5.jpg]]<br />
<br />
<br />
[[Bologna#Model | Back]]</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T14:48:36Z<p>Francesca.bugane2: /* Model */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
Knowing that the β functions represent: <br />
:: :glucose dependent LacY transcription inhibitor; <br />
:: :dependent LacY transcription enhancer;<br />
:: :glucose dependent IPTG uptake inhibitor;<br />
:: :IPTG dependent IPTG uptake enhancer<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp2.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/IB| Iros Barozzi]] <br />
:'''Graduate Student''' <br />
::[[Bologna_University/AP | Alice Pasini]] <br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
:: Sara Montagna • [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Beta4.jpgFile:Beta4.jpg2007-10-26T14:43:58Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Beta3.jpgFile:Beta3.jpg2007-10-26T14:43:07Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Beta2.jpgFile:Beta2.jpg2007-10-26T14:42:11Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/File:Beta1.jpgFile:Beta1.jpg2007-10-26T14:40:53Z<p>Francesca.bugane2: </p>
<hr />
<div></div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T14:38:15Z<p>Francesca.bugane2: /* Model */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/ST | Silvia Tamarri]], [[Bologna_University/FB | Francesca Buganè]] and [[Bologna_University/MM| Michela Mirri]] undergraduated students in Biomedical Engineering; [[Bologna_University/FP | Francesco Pasqualini]], graduated student in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], graduated student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], graduated student in Industrial Biotechnology and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronic. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], graduated in Pharmaceutical Biotechnologies and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | gag<br />
| style="background:white;" | djak<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | asih<br />
| style="background:white;" | lhsld<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | asjbdb<br />
| style="background:white;" | sgakdg<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | asjbdb<br />
| style="background:white;" | sgakdg<br />
|-<br />
|}<br />
<br />
<br />
Knowing that the β functions represent: <br />
:: :glucose dependent LacY transcription inhibitor; <br />
:: :dependent LacY transcription enhancer;<br />
:: :glucose dependent IPTG uptake inhibitor;<br />
:: :IPTG dependent IPTG uptake enhancer<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp2.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/IB| Iros Barozzi]] <br />
:'''Graduate Student''' <br />
::[[Bologna_University/AP | Alice Pasini]] <br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
:: Sara Montagna • [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T14:37:59Z<p>Francesca.bugane2: /* Model */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/ST | Silvia Tamarri]], [[Bologna_University/FB | Francesca Buganè]] and [[Bologna_University/MM| Michela Mirri]] undergraduated students in Biomedical Engineering; [[Bologna_University/FP | Francesco Pasqualini]], graduated student in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], graduated student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], graduated student in Industrial Biotechnology and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronic. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], graduated in Pharmaceutical Biotechnologies and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[[[Image:Beta1.jpg]]<br />
| style="background:white;" | gag<br />
| style="background:white;" | djak<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | asih<br />
| style="background:white;" | lhsld<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | asjbdb<br />
| style="background:white;" | sgakdg<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | asjbdb<br />
| style="background:white;" | sgakdg<br />
|-<br />
|}<br />
<br />
<br />
Knowing that the β functions represent: <br />
:: :glucose dependent LacY transcription inhibitor; <br />
:: :dependent LacY transcription enhancer;<br />
:: :glucose dependent IPTG uptake inhibitor;<br />
:: :IPTG dependent IPTG uptake enhancer<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp2.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/IB| Iros Barozzi]] <br />
:'''Graduate Student''' <br />
::[[Bologna_University/AP | Alice Pasini]] <br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
:: Sara Montagna • [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T14:36:15Z<p>Francesca.bugane2: /* Model */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/ST | Silvia Tamarri]], [[Bologna_University/FB | Francesca Buganè]] and [[Bologna_University/MM| Michela Mirri]] undergraduated students in Biomedical Engineering; [[Bologna_University/FP | Francesco Pasqualini]], graduated student in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], graduated student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], graduated student in Industrial Biotechnology and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronic. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], graduated in Pharmaceutical Biotechnologies and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | sfajg<br />
| style="background:white;" | gag<br />
| style="background:white;" | djak<br />
|-<br />
! style="background:white;" | asjdks<br />
| style="background:white;" | asih<br />
| style="background:white;" | lhsld<br />
|-<br />
! style="background:white;" | agk<br />
| style="background:white;" | asjbdb<br />
| style="background:white;" | sgakdg<br />
|-<br />
! style="background:white;" | agk<br />
| style="background:white;" | asjbdb<br />
| style="background:white;" | sgakdg<br />
|-<br />
|}<br />
<br />
<br />
Knowing that the β functions represent: <br />
:: :glucose dependent LacY transcription inhibitor; <br />
:: :dependent LacY transcription enhancer;<br />
:: :glucose dependent IPTG uptake inhibitor;<br />
:: :IPTG dependent IPTG uptake enhancer<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp2.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/IB| Iros Barozzi]] <br />
:'''Graduate Student''' <br />
::[[Bologna_University/AP | Alice Pasini]] <br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
:: Sara Montagna • [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T14:34:00Z<p>Francesca.bugane2: /* Model */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/ST | Silvia Tamarri]], [[Bologna_University/FB | Francesca Buganè]] and [[Bologna_University/MM| Michela Mirri]] undergraduated students on Biomedical Engineering; [[Bologna_University/FP | Francesco Pasqualini]], graduated student in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], graduated student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], graduated student in Industrial Biotechnology and [[Bologna_University/AP | Alice Pasini]], a PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronic. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], graduated in Pharmaceutical Biotechnologies and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main characteristics of this device is to be a “smart” switch: that means switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the modification of the value in some quantities that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technic applications since it solves the instability problems, in fact, the minimal variation that cause output changes must be as large as the difference between the two thresholds. Then noise is not so critical.<br />
<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.''Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature control the heater that determines the temperature), it is able to automatically mantaine a warm temperature: and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit tries to riproduce this fondamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
We wanted to reproduce the same principle with a genetic circuit. To do so we exploited one of such system, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, E.''coli'' can either survive by metabolizing glucose or, in case glucose is missing, lactose. Lactose or glucose metabolism-modes are the two stable state of our system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. That is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] is constitutively repressed thank to the presence of the LacI protein that inhibits the transcription of the genes downstream of the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When there is IPTG in the environment of the cell, several concurrent processes take place. IPTG enters through the membrane and after some processing it is able to remove the repressor LacI, thus allowing the transcription of genes LacY that in turn enhance the IPTG uptake and increase its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the switch.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
The second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the information we had on the Schmitt Trigger ([[#Introduction |See in the introducion]]) and on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon], we decided to explore the behavior of our genetic circuit by mathematically modeling the parts depicted in Figure 4.<br />
<br />
[[Image:modellomath1.jpg|center]]<br />
<br />
Figure 6. ''The core of our circuit: the pLac promoter, inhibited by protein LacI, and genes LacY and GFP the positive feedback input and reporter gene respectively.''<br />
<br />
<br />
===Model===<br />
In the following we describe the 3 differential equations we used in the model. They represent the variation over time (derivative d/dt) of the production of the mRNA of lacY (called T for transcript), of the production of the protein LacY (called P for protein) and the variation of the input to our circuit IPTG_in. These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex to avoid [[Bologna University/Equations mess | this]].<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | sfajg<br />
| style="background:white;" | gag<br />
| style="background:white;" | djak<br />
|-<br />
! style="background:white;" | asjdks<br />
| style="background:white;" | asih<br />
| style="background:white;" | lhsld<br />
|-<br />
! style="background:white;" | agk<br />
| style="background:white;" | asjbdb<br />
| style="background:white;" | sgakdg<br />
|-<br />
|}<br />
<br />
Knowing that the β functions represent: <br />
:: :glucose dependent LacY transcription inhibitor; <br />
:: :dependent LacY transcription enhancer;<br />
:: :glucose dependent IPTG uptake inhibitor;<br />
:: :IPTG dependent IPTG uptake enhancer<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP, this is simply:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the parameters are defined [[Bologna University/here | here]].<br />
<br />
To simulate the evolution of these variables we used [[Bologna University/simulink | simulink]].<br />
<br />
===Simulation===<br />
With this model, we can predict the circuit behavior and assess if it actually has bistability and [[Bologna University/Hysteresis | hysteresis]]. In the following we show the results of the simulations.<br />
<br />
<br />
[[Image:Iptg_Gfp2.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
<br />
<br />
[[Image:Stability_plane1.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds values change for varying levels of external glucose.'' <br />
''All the points that lie between the two curves represent unstable states, the system will not remain in that state but it will move to a state in one of the two other areas.''<br />
<br />
<br />
<br />
<br />
[[Image:grafico2.jpg|center]]<br />
<br />
Figure 9.''Shows how the fully induced steady state depend on the External Glucose.''<br />
<br />
==Image Acquisition and Elaboration==<br />
<br />
===Experimental SetUp===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10.<br />
<br />
<br />
A schematic design of our acquisition system based on the fluorescence microscope is shown in figure.<br />
<br />
The illumination system is composed of a 75 Watt Xenon arc lamp connected to a Photon Technology Instruments DeltaRAM X monochromator, which breaks up a single polychromatic light beam into several monochromatic light beams (with only one wavelength each). Only the selected wavelength can pass through the output port and reach the microscope.<br />
<br />
The system’s core is a Nikon Eclipse TE2000-U inverted fluorescence microscope. For GFP image acquisition we used a B-2A filter by Nikon with an excitation band between 450 and 490 nm and the optimal emission placed at 520 nm. <br />
<br />
The camera used to acquire images and film segments is a Nikon DS-5m with a DS-U1 controller. This one receives the acquired signal form the camera through a serial connection and sends it to the PC through an USB slot. Nikon also supplied an interface software for image acquisition and elaboration.<br />
<br />
The Photon Technology Instruments PMT 814 photomultiplier tube is connected to the microscope’s left port, on which the whole light signal can be deviated. Acquisition can be implemented in two different modes, depending on the signal amplitude.<br />
<br />
The control software is implemented in a Labview environment and permits the regulation of the excitation wavelength and the calibration of the system. It also pictures the output signal from the photomultiplier, which can be memorized and elaborated.<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up illustrated [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here], we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
[[Image:orig100.jpg|left]] <br />
<br />
[[Image:orig200.jpg|absright]]<br />
<br />
Figure 11.<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with our segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:segm100.jpg|left]]<br />
<br />
[[Image:segm200.jpg|absright]]<br />
<br />
Figure 12.<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
-''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
-''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
:1.<u>pTeTR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for LacI gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTeTR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTeTR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of LacI gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and cI, LacY and GFP genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and PstI enzymes.<br />
<br />
=Acknowledgments=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/IB| Iros Barozzi]] <br />
:'''Graduate Student''' <br />
::[[Bologna_University/AP | Alice Pasini]] <br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
:: Sara Montagna • [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image Acquisition | Image Acquisition and Elaboration]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University/Fluorescence reliance on Promoter and Medium | Fluorescence reliance on Promoter and Medium]]<br />
:[[Bologna_University/Conclusions | '''Conclusions''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Francesca.bugane2