http://2007.igem.org/wiki/index.php?title=Special:Contributions/Skundu&feed=atom&limit=50&target=Skundu&year=&month=2007.igem.org - User contributions [en]2024-03-28T21:23:39ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/The_CompanyThe Company2007-09-20T14:17:46Z<p>Skundu: /* '''The Company''' */</p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{| width="35%"<br />
|-<br />
|align="center"|<br />
<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|-<br />
|align="center"|<br />
'''The Bangalore iGEM Team, 07'''<br />
<br />
<br />
<br />
<br />
<br />
<br />
|}<br />
<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
! align="center"|[[Bangalore|Bangalore]]<br />
! align="center"|[[The Company|The Team]]<br />
! align="center"|[[The Mission|The Mission]]<br />
! align="center"|[[Experiments|Experiments]]<br />
! align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
== '''The Company''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
[[Image:Ncbs_Logo.jpg|200px|center]]<br />
<br />
<br />
<br />
<br />
<br />
*'''Mukund''': Chief Mentor, Consultant and The Godfather<br />
*'''Sugat''': Right-hand Man and Manager of Operations<br />
*'''Nilesh''': The Man for all Seasons <br />
*'''Vivek''': Chief Troubleshooter, often his role is confused with one of a Troublemaker <br />
*'''Krishna''': The Sharpshooter <br />
*'''Navneet''': Quorum Sensor(ed) <br />
*Senthil: The Wikiman, also fondly referred to as the Bio Info God<br />
*'''[[User:Skundu|Shashanka]]''': The sili'con' man<br />
*'''[[User:Smore|Sushant]]''': Master Linguist, also called Chow-How Yaake<br />
*'''Varun''': The Voice of Reason and Anti-Reason<br />
*'''Vini''': The Pooh<br />
*'''''Escherichia coli'' K12Z1''': The Workhorse<br />
|<br />
<gallery><br />
Image:Mukund.jpg|Mukund Thattai<br />
Image:Sugat.jpg|Sugat Dhabolkar<br />
Image:Nilesh.jpg|Nilesh Aghera<br />
Image:Vivek.jpg|S. Vivek Raj<br />
<br />
Image:KrishnaR.JPG|Krishna Ramkumar<br />
Image:Navneet.jpg|Navneet Rai<br />
Image:Senthil.jpg|Senthil Kumar<br />
Image:Shashank.jpg|Shashanka S. Kundu<br />
<br />
Image:Sushant.jpg|Sushant More<br />
Image:Varun.jpg|Varun Sreenivasan<br />
Image:Vini.jpg|Vini Gautam<br />
Image:K12Z1.jpg|''E.coli'' K12Z1<br />
</gallery><br />
|}</div>Skunduhttp://2007.igem.org/wiki/index.php/June_7June 72007-08-25T14:34:33Z<p>Skundu: </p>
<hr />
<div><br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''</div>Skunduhttp://2007.igem.org/wiki/index.php/June_6June 62007-08-25T14:34:25Z<p>Skundu: </p>
<hr />
<div><br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''</div>Skunduhttp://2007.igem.org/wiki/index.php/July_10July 102007-08-25T14:33:52Z<p>Skundu: </p>
<hr />
<div><br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''</div>Skunduhttp://2007.igem.org/wiki/index.php/July_11July 112007-08-25T14:33:44Z<p>Skundu: </p>
<hr />
<div><br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''</div>Skunduhttp://2007.igem.org/wiki/index.php/July_12July 122007-08-25T14:33:35Z<p>Skundu: </p>
<hr />
<div><br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''</div>Skunduhttp://2007.igem.org/wiki/index.php/July_15July 152007-08-25T14:33:14Z<p>Skundu: </p>
<hr />
<div><br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''</div>Skunduhttp://2007.igem.org/wiki/index.php/July_13July 132007-08-25T14:33:02Z<p>Skundu: </p>
<hr />
<div><br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''</div>Skunduhttp://2007.igem.org/wiki/index.php/July_15July 152007-08-25T14:32:49Z<p>Skundu: </p>
<hr />
<div>'''Presentation Day'''<br />
<br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-24T15:51:56Z<p>Skundu: /* Experiment */</p>
<hr />
<div>{| cellpadding="0" cellspacing="0"<br />
|- width="37%" <br />
|align="center"|<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|rowspan="3" width="62%" |[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
|- <br />
|align="center"|'''The NCBS iGEM 2007 Experiments'''<br />
|-<br />
| align="justify"|<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
!align="center"|[[Bangalore|Bangalore]]<br />
!align="center"|[[The Company|The Team]]<br />
!align="center"|[[The Mission|The Mission]]<br />
!align="center"|[[Experiments|Experiments]]<br />
!align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
{|<br />
|- align="justify"<br />
|[[Image:pLC_dup.png|400px]]<br />
|Details...<br />
|}<br />
<br />
*pT.luxI.Cfp<br />
{|<br />
|- align="justify"<br />
|[[Image:pTIC_dup.png|400px]]<br />
|Details...<br />
|}<br />
<br />
*pL.luxI.Cfp<br />
{|<br />
|- align="justify"<br />
|[[Image:pLIC_dup.png|400px]]<br />
|Details...<br />
|}<br />
<br />
*pL.luxR.Yfp<br />
{|<br />
|- align="justify"<br />
|[[Image:pLRY_dup.png|400px]]<br />
|Details...<br />
|}<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
{|<br />
|- align="justify"<br />
|[[Image:Openloop_0.png|400px]]<br />
|Details...<br />
|}<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
{|<br />
|- align="justify"<br />
|[[Image:Openloop_1.png|400px]]<br />
|Details...<br />
|}<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
{|<br />
|- align="justify"<br />
|[[Image:Openloop_5.png|400px]]<br />
|Details...<br />
|}<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
{|<br />
|- align="justify"<br />
|[[Image:Openloop_10.png|400px]]<br />
|Details...<br />
|}<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
{|<br />
|- align="justify"<br />
|[[Image:Openloop_20.png|400px]]<br />
|Details...<br />
|}<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
{|<br />
|- align="justify"<br />
|[[Image:Openloop_50.png|400px]]<br />
|Details...<br />
|}<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-24T15:48:29Z<p>Skundu: /* Experiment */</p>
<hr />
<div>{| cellpadding="0" cellspacing="0"<br />
|- width="37%" <br />
|align="center"|<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|rowspan="3" width="62%" |[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
|- <br />
|align="center"|'''The NCBS iGEM 2007 Experiments'''<br />
|-<br />
| align="justify"|<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
!align="center"|[[Bangalore|Bangalore]]<br />
!align="center"|[[The Company|The Team]]<br />
!align="center"|[[The Mission|The Mission]]<br />
!align="center"|[[Experiments|Experiments]]<br />
!align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
[[Image:pLC_dup.png|400px]]<br />
<br />
*pT.luxI.Cfp<br />
[[Image:pTIC_dup.png|400px]]<br />
<br />
*pL.luxI.Cfp<br />
[[Image:pLIC_dup.png|400px]]<br />
<br />
*pL.luxR.Yfp<br />
[[Image:pLRY_dup.png|400px]]<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
[[Image:Openloop_0.png|400px]]<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
[[Image:Openloop_1.png|400px]]<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
[[Image:Openloop_5.png|400px]]<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
[[Image:Openloop_10.png|400px]]<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
[[Image:Openloop_20.png|400px]]<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
[[Image:Openloop_50.png|400px]]<br />
<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-24T15:47:19Z<p>Skundu: /* Experiment */</p>
<hr />
<div>{| cellpadding="0" cellspacing="0"<br />
|- width="37%" <br />
|align="center"|<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|rowspan="3" width="62%" |[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
|- <br />
|align="center"|'''The NCBS iGEM 2007 Experiments'''<br />
|-<br />
| align="justify"|<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
!align="center"|[[Bangalore|Bangalore]]<br />
!align="center"|[[The Company|The Team]]<br />
!align="center"|[[The Mission|The Mission]]<br />
!align="center"|[[Experiments|Experiments]]<br />
!align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
[[Image:pLC_dup.png|400px]]<br />
*pT.luxI.Cfp<br />
[[Image:pTIC_dup.png|400px]]<br />
*pL.luxI.Cfp<br />
[[Image:pLIC_dup.png|400px]]<br />
*pL.luxR.Yfp<br />
[[Image:pLRY_dup.png|400px]]<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
[[Image:Openloop_0.png|400px]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
[[Image:Openloop_1.png|400px]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
[[Image:Openloop_5.png|400px]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
[[Image:Openloop_10.png|400px]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
[[Image:Openloop_20.png|400px]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
[[Image:Openloop_50.png|400px]]<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/File:Openloop_50.pngFile:Openloop 50.png2007-08-24T15:46:38Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/File:Openloop_20.pngFile:Openloop 20.png2007-08-24T15:46:24Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/File:Openloop_10.pngFile:Openloop 10.png2007-08-24T15:46:09Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/File:Openloop_5.pngFile:Openloop 5.png2007-08-24T15:45:50Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/File:Openloop_1.pngFile:Openloop 1.png2007-08-24T15:45:27Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/File:Openloop_0.pngFile:Openloop 0.png2007-08-24T15:45:11Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-24T15:45:01Z<p>Skundu: /* Open loops */</p>
<hr />
<div>{| cellpadding="0" cellspacing="0"<br />
|- width="37%" <br />
|align="center"|<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|rowspan="3" width="62%" |[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
|- <br />
|align="center"|'''The NCBS iGEM 2007 Experiments'''<br />
|-<br />
| align="justify"|<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
!align="center"|[[Bangalore|Bangalore]]<br />
!align="center"|[[The Company|The Team]]<br />
!align="center"|[[The Mission|The Mission]]<br />
!align="center"|[[Experiments|Experiments]]<br />
!align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
[[Image:pLC_dup.png]]<br />
*pT.luxI.Cfp<br />
[[Image:pTIC_dup.png]]<br />
*pL.luxI.Cfp<br />
[[Image:pLIC_dup.png]]<br />
*pL.luxR.Yfp<br />
[[Image:pLRY_dup.png]]<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
[[Image:Openloop_0.png]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
[[Image:Openloop_1.png]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
[[Image:Openloop_5.png]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
[[Image:Openloop_10.png]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
[[Image:Openloop_20.png]]<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
[[Image:Openloop_50.png]]<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/File:PLRY_dup.pngFile:PLRY dup.png2007-08-24T15:44:00Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/File:PLIC_dup.pngFile:PLIC dup.png2007-08-24T15:43:42Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/File:PTIC_dup.pngFile:PTIC dup.png2007-08-24T15:43:20Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/File:PLC_dup.pngFile:PLC dup.png2007-08-24T15:42:55Z<p>Skundu: </p>
<hr />
<div></div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-24T15:35:46Z<p>Skundu: /* Equivalences */</p>
<hr />
<div>{| cellpadding="0" cellspacing="0"<br />
|- width="37%" <br />
|align="center"|<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|rowspan="3" width="62%" |[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
|- <br />
|align="center"|'''The NCBS iGEM 2007 Experiments'''<br />
|-<br />
| align="justify"|<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
!align="center"|[[Bangalore|Bangalore]]<br />
!align="center"|[[The Company|The Team]]<br />
!align="center"|[[The Mission|The Mission]]<br />
!align="center"|[[Experiments|Experiments]]<br />
!align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
[[Image:pLC_dup.png]]<br />
*pT.luxI.Cfp<br />
[[Image:pTIC_dup.png]]<br />
*pL.luxI.Cfp<br />
[[Image:pLIC_dup.png]]<br />
*pL.luxR.Yfp<br />
[[Image:pLRY_dup.png]]<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/July_6July 62007-08-18T15:22:07Z<p>Skundu: /* Analysis */</p>
<hr />
<div>Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''<br />
<br />
==Experiments==<br />
<br />
<br />
* Open Loop [Ee]<br />
<br />
i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=50 ng/mL and inoculum=1,0.1 uL/mL<br />
<br />
ii) K12Z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=50 ng/mL and inoculum=1,0.1 uL/mL<br />
<br />
iii) pL.LuxR.Y.pR.C, K12z1, pL.Y were inoculated in LB<br />
<br />
iv) The contents of the flask with OD=0.217 were filtered out.<br />
<br />
v) 7 dilutions were made (full range of IPTG conc) for pL.LuxR.Y.pR.C with AI from 50 ng/mL aTc and new 2xM9 in 1:1 ratio.<br />
<br />
<br />
* Open Loop [Cc]<br />
<br />
i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=5 ng/mL and inoculum=1,0.1 uL/mL<br />
<br />
ii) K12z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=5 ng/mL and inoculum=1,0.1 uL/mL<br />
<br />
iii) pL.LuxR.Y.pR.C, K12z1, pL.Y were inoculated in LB.<br />
<br />
iv) The contents of the flask with OD=0.103 were filtered out.<br />
<br />
v) 7 dilutions over the full range of IPTG were made for pL.LuxR.Y.pR.C with AI from 5 ng/mL aTc and new 2xM9 in 1:1 ratio<br />
<br />
<br />
* Control experiments:<br />
** -ve control: K12z1<br />
**Inoculated positive control: <br />
***pL.YFP; 1xGlu M9; 500 uM IPTG<br />
***I726081; K12Z1 in aTc (50 ng/mL) medium and new 2xGlu M9 in 1:1 ratio; 500 uM IPTG<br />
***I726081; only new 1xGlu M9; 500 uM IPTG<br />
<br />
<br />
'''Microscopy'''<br />
<br />
* The open loop samples [Ee] were imaged and FACS was done.<br />
<br />
* The open loop samples [Cc] were imaged and FACS was done.<br />
<br />
* The open loop samples [Dd] were imaged and FACS was done.<br />
<br />
==Analysis==<br />
<br />
Today we compiled the data collected from all our open-loop experiments performed at various concentrations of aTc (ng/ml).<br />
The following matrix of information was obtained:<br />
{|cellspacing="0" cellpadding="3" border="1" align="left"<br />
|-align="center"<br />
!colspan="2" rowspan="2"|<br />
CFP mean<br />
<br />
values<br />
|colspan="7"|<br />
IPTG (uM) or luxR.YFP<br />
|-align="center"<br />
|0 <br />
|5 <br />
|10<br />
|50<br />
|100<br />
|500<br />
|1000<br />
|-align="center"<br />
|rowspan="6"|<br />
aTc (ng/ml)<br />
<br />
or<br />
<br />
luxI.CFP<br />
|0<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|1<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|5<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|10<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|20<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|50<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|}<br />
{|cellspacing="0" cellpadding="3" border="1" align="right"<br />
|-align="center"<br />
!colspan="2" rowspan="2"|<br />
YFP mean<br />
<br />
values<br />
|colspan="7"|<br />
IPTG (uM) or luxR.YFP<br />
|-align="center"<br />
|0 <br />
|5 <br />
|10<br />
|50<br />
|100<br />
|500<br />
|1000<br />
|-align="center"<br />
|rowspan="6"|<br />
aTc (ng/ml)<br />
<br />
or<br />
<br />
luxI.CFP<br />
|0<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|1<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|5<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|10<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|20<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|-align="center"<br />
|50<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|<br />
|}<br />
{|align="center" width="37%"<br />
|-<br />
|align="justify"|These values, obtained from the microscopy experiments, give us a clear idea of the equivalence between luxI.CFP and luxI.YFP.<br />
<br />
A scatter plot with each dot representing a cell was also plotted to see the variation of fluorescence with the change in aTc and IPTG concentrations.<br />
{|align="center"<br />
|-<br />
|<br />
*<font color="Red">Red</font> : 0 ng/ml aTc<br />
|<br />
*<font color="Green">Green</font> : 1 ng/ml aTc<br />
|-<br />
|<br />
*<font color="Blue">Blue</font> : 5 ng/ml aTc<br />
|<br />
*<font color="Cyan">Cyan</font> : 10 ng/ml aTc<br />
|-<br />
|<br />
*<font color="Magenta">Magenta</font> : 20 ng/ml aTc<br />
|<br />
*<font color="Yellow">Yellow</font> : 50 ng/ml aTc<br />
|}<br />
|}<br />
<br />
<br />
{|align="center" width="85%"<br />
!align="center"|CFP Fluorescence<br />
!align="center"|YFP Fluorescence<br />
|-<br />
|[[Image:Openloop_cfp_jul6.jpg|center|thumb|350px|CFP fluorescence]]<br />
|[[Image:Openloop_yfp_jul6.jpg|center|thumb|350px|YFP fluorescence]]<br />
|-<br />
|[[Image:surf_plot_cfp_jul6.jpg|center|thumb|350px|CFP fluorescence]]<br />
|[[Image:surf_plot_yfp_jul6.jpg|center|thumb|350px|YFP fluorescence]]<br />
|-<br />
|colspan="2" align="justify"|<br />
Surface plots : The increasing values along the luxI.CFP axis are the increasing concentrations of aTc whereas the increasing values along the luxR.YFP axis are the increasing concentrations of IPTG.<br />
|}</div>Skunduhttp://2007.igem.org/wiki/index.php/July_14July 142007-08-14T10:17:11Z<p>Skundu: </p>
<hr />
<div>'''Presentation Day'''<br />
<br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''<br />
<br />
==Experiments==<br />
<br />
== Analysis ==<br />
<br />
== Discussion ==</div>Skunduhttp://2007.igem.org/wiki/index.php/July_14July 142007-08-14T10:16:59Z<p>Skundu: </p>
<hr />
<div>'''Presentation Day'''<br />
Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''<br />
<br />
==Experiments==<br />
<br />
== Analysis ==<br />
<br />
== Discussion ==</div>Skunduhttp://2007.igem.org/wiki/index.php/July_14July 142007-08-14T10:15:10Z<p>Skundu: </p>
<hr />
<div>'''Presentation Day'''</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-14T10:06:54Z<p>Skundu: </p>
<hr />
<div>{| cellpadding="0" cellspacing="0"<br />
|- width="37%" <br />
|align="center"|<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|rowspan="3" width="62%" |[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
|- <br />
|align="center"|'''The NCBS iGEM 2007 Experiments'''<br />
|-<br />
| align="justify"|<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
!align="center"|[[Bangalore|Bangalore]]<br />
!align="center"|[[The Company|The Team]]<br />
!align="center"|[[The Mission|The Mission]]<br />
!align="center"|[[Experiments|Experiments]]<br />
!align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
*pT.luxI.Cfp<br />
*pL.luxI.Cfp<br />
*pL.luxR.Yfp<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/E-NotebookE-Notebook2007-08-14T10:05:25Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{| width="37%"<br />
|-<br />
|align="center"|<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|- <br />
|align="center"|'''The Bangalore iGEM Journal, 07'''<br />
<br />
<br />
<br />
<br />
|}<br />
<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
!align="center"|[[Bangalore|Bangalore]]<br />
!align="center"|[[The Company|The Team]]<br />
!align="center"|[[The Mission|The Mission]]<br />
!align="center"|[[Experiments|Experiments]]<br />
!align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
<br />
==e-Notebook == <br />
{| align="center" border="0" cellpadding="5" cellspacing="3" <br />
!colspan="7"|June 07 <br />
!colspan="7"| <br />
!colspan="7"|July 07 <br />
|-<br />
|'''S''' <br />
|M <br />
|T <br />
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|F <br />
|S <br />
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|'''S''' <br />
|M <br />
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|T <br />
|F <br />
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|[[June 1|1]] <br />
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|[[July 3|3]] <br />
|[[July 4|4]] <br />
|[[July 5|5]] <br />
|[[July 6|6]] <br />
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|3 <br />
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|9 <br />
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|[[July 10|10]] <br />
|[[July 11|11]] <br />
|[[July 12|12]] <br />
|[[July 13|13]] <br />
|[[July 14|14]] <br />
|-<br />
|10 <br />
|[[June 11|11]] <br />
|[[June 12|12]] <br />
|[[June 13|13]] <br />
|[[June 14|14]] <br />
|[[June 15|15]] <br />
|[[June 16|16]] <br />
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|[[June 17|17]] <br />
|[[June 18|18]] <br />
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|[[June 21|21]] <br />
|[[June 22|22]] <br />
|[[June 23|23]] <br />
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|- <br />
|[[June 24|24]] <br />
|[[June 25|25]] <br />
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|[[June 28|28]] <br />
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|}<br />
<br />
=='''Protocols''' ==<br />
<br />
'''1) Induction experiments:'''<br />
Grow in LB for 10 hours<br />
Then, induce in Glu M9 for 12 hours at various concns of IPTG/aTc<br />
+ve control: pL YFP in 50,100 uM IPTG <br />
-ve control: K12z1 <br />
Take microscopy images<br />
<br />
'''2) Open loop experiments:'''<br />
i) Morning imaging (followed usually)<br />
Day 0: 0000: Inoculate pTLuxI.C in LB<br />
Day 1: 0100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
1230: Inoculate pLLuxR.Y pRC in LB<br />
2200: Filter required OD flask<br />
2230: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 <br />
Day 2: 1000: Take microscopy images<br />
ii) Night imaging<br />
Day 1: 1100: Inoculate pTLuxI.C in LB<br />
2100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
2330: Inoculate pLLuxR.YpRC in LB<br />
Day 2: 0900: Filter required OD flask<br />
0930: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 in 50 old:50 new 2xM9<br />
2130: Take microscopy images<br />
<br />
==Notes==<br />
*All inductions with IPTG: 0,5,10,50,100,500,1000 uM<br />
<br />
*All inductions with aTc: 0,1,5,10,20,50 ng/mL<br />
<br />
*The open loop experiments to be carried out with following aTc concentrations:<br />
<br />
[A] : 20 ng/ml<br />
<br />
[B] : 10 ng/ml<br />
<br />
[C] : 5 ng/ml<br />
<br />
[D] : 1 ng/ml<br />
<br />
[E] : 50 ng/ml<br />
<br />
[F] : 0 ng/ml<br />
<br />
[AA] : 20 ng/ml (repeat A)<br />
<br />
[FF] : 0 ng/ml (repeat F)<br />
<br />
<br />
The '''''DUPLICATES''''' for each of the above experiments are denoted by [Aa], [Bb], [Cc], [Dd], [Ee] and [Ff] respectively.<br />
<br />
== This Week ==<br />
<br />
{|-<br />
|<br />
Mon, July 2: pL LuxI.C for IPTG-aTc equivalence (morn)<br />
Tue, July 3: pL LuxR.Y for cfp-yfp equivalence, <br />
pT LuxI.C for IPTG-aTc<br />
Wed, July 4: Open loop [E],[F] (aTc) <br />
At OD 0.1: To prove scaling argument for AI.<br />
Thu, July 5: aTC 10,20 ng/mL [X,Y]<br />
DUPLICATES:<br />
---------<br />
At OD 0.2: <br />
Fri, July 6: Open loop: aTC 20,10 ng/mL [A',B'] <br />
Sat, July 7: Open loop: aTC 5,1 ng/mL [C',D']<br />
|}<br />
<br />
<br />
<br />
== Experiments Record ==<br />
*Calibration of the number of colonies obtained at different optical densities.<br />
*Transformation of competent E.coli (strain K12Z1) cells with constructs.<br />
*'''Induction of the construct pLac cfp''' <br />
E.coli cells transformed with the construct pLac cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac cfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
Cells were allowed to grow till the optical density was in the range of 0.05-0.1 (early exponential phase) and were imaged using a phase contrast microscope.<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pLac yfp'''<br />
E.coli cells transformed with the construct pLac yfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac yfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of YFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pTet.luxI.cfp''' <br />
E.coli cells transformed with the construct pTet.luxI.cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of aTc. A stock concentration of 1mg/ml was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!10ug/ml aTc (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pTet luxI cfp(0 ng/ml)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(1 ng/ml)<br />
|0.3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(10 ng/ml)<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(25 ng/ml)<br />
|7.5 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(50 ng/ml)<br />
|15 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(100 ng/ml)<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of aTc.<br />
<br />
*'''Open Loop Trial'''<br />
E. coli cells containing pTet.luxI.cells were first cultured in Luria Bertani medium for 10 hours following which they were transferred to M9 medium where they were induced with aTc (50 ng/ml). The cells were then allowed to grow in M9 for 12 hours.<br />
<br />
The medium (now containing the autoinducer) was filtered and was added to an equal volume of freshly prepared M9 medium containing twice the concentration of glucose. E.coli cells containing pLac.luxR.yfp.pR.cfp (previously grown in LB medium) were inoculated into the M9 medium prepared and were induced with different concentrations of IPTG. Cells were allowed to grow for 12 hours.<br />
<br />
*'''The Scaling Argument'''<br />
<br />
Two sets of experiments would be carried out, where cells would be grown to an optical density of 0.257 & 0.015 respectively in order to check for density dependence of autoinducer production. The supernatent would be filtered out and the one from the OD=0.257 would be scaled down to OD=0.015. The cells would then be imaged using a phase contrast microscope. The scaling argument would be proved if we get the same cfp expression from both.<br />
<br />
== Discussions ==</div>Skunduhttp://2007.igem.org/wiki/index.php/The_MissionThe Mission2007-08-14T09:24:03Z<p>Skundu: </p>
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<br />
== '''The Objective''' ==<br />
<br />
To investigate multistability and hysteresis in combinatorially constructed synthetic ''Vibrio'' quorum sensing circuits<br />
<br />
== '''The Target - ''Vibrio'' Quorum Sensing System''' ==<br />
<br />
[[Image:vibrio_qs.jpg|thumb|200px|Fig. 1: Quorum sensing in Vibrio fischeri - a LuxI-R signalling circuit. Red triangles indicate the autoinducer that is produced by LuxI. OM, outer membrane; IM, inner membrane.]]<br />
<br />
Quorum sensing is a phenomenon by which bacteria sense a critical cell density before turning on the expression of certain genes. It involves the gradual build-up of a chemical termed the 'autoinducer' in the cell. The autoinducer freely diffuses across the cell membrane and hence, its concentration is population density dependent. When the concentration crosses a threshold, the bacteria switch to a different physiological state such as bioluminescence, virulent gene expression, and bio-film formation. <br />
<br />
In ''Vibrio fischeri'', when the population density (and hence the concentration of autoinducer) crosses a certain threshold, the expression of a set of genes that is required for bioluminescence is turned on. The production of the autoinducer is under the control of a gene, the expression of which involves ''positive feedback''. Figure 1 shows the various components of this system (Ref. 1).<br />
<br />
View a powerpoint presentation. [[Media:QSppt.ppt|Click here.]]<br />
<br />
==='''Why did we pick this system?'''===<br />
<br />
The Vibrio quorum sensing system involves a well-defined set of genes and a promoter, and has a degree of complexity that offers wide scope for exploration. Additionally, the concentration of the active transcriptional regulator, LuxR* is dependent on 3 factors:<br />
i) The concentration of LuxI <br />
ii) The population density <br />
iii) The concentration of LuxR<br />
<br />
All these factors can be experimentally controlled. Thus, the nature of the system offers one an extremely good handle on the feedback strength of the genetic circuit.<br />
<br />
=='''A note on Multistability and Hysteresis'''==<br />
[[Image:Break_feedback_loop.jpg|thumb|200px|left|Fig. 2: Schematic view of a feedback system before (Left) and after (Right) breaking the feedback loop. ‘ω’ is the input of the open-loop system and ‘η’ is the output.]]<br />
<br />
In the recent past, multistability has been an important recurring theme in studies on cell signalling. Angeli et al (Ref. 2) have shown that for a class of feedback systems of arbitrary order, the stability properties and bifurcation diagram of the system can be deduced mathematically from how the system behaves when feedback is blocked. [[Image:Io_curve.jpg|thumb|200px|right|Fig. 3: Steady state I/O static characteristic curve for the open loop of the mutually inhibitory Cdc2-Cyclin B/Wee1 feedback system (red). The solid blue line represents η as a function of ω for unitary feedback. ]]The system is guaranteed to be bistable for some range of feedback strengths provided the feedback-blocked system is monotone and shows a sigmoidal characteristic. <br />
<br />
A simple graphical method can be used to deduce the stability behaviour of such systems (Ref. 2). The key feature of this approach is to view the positive feedback system as a feedback closure of its corresponding 'open loop' system. This open loop system is obtained by breaking the feedback loop at the point of feedback (Fig. 2).<br />
<br />
Now, one can experimentally manipulate the amount of input (ω) and monitor the output (η) as a function of ω. The fixed points of the corresponding closed loop system are then obtained by intersecting η=f(ω) with the straight line, η=(1/ν)ω, where ' ν ' is the feedback strength. At these points of intersection, the open loop system exactly mimics the closed loop system. As shown in Fig. 3, they represent two stable steady states (I and III) and one unstable steady state (II).<br />
<br />
[[Image:Bifurcation.jpg|thumb|185px|left|Fig. 4: Bifurcation diagram, showing bistability when the feedback strength ‘ν’ is between ~0.83 and ~1.8. ]]<br />
<br />
All positive-feedback, multistable systems show the associated property of ''hysteresis''. This can be explained by analysing the corresponding bifurcation diagram, which is a plot of the steady states as a function of the feedback strength (see Fig. 4). To do this, one can vary the feedback strength, ‘ν’ and find the different points at which the I/O characteristic curve intersects the equivalence line η=(1/ν)ω. At high and low feedback strength values, the system is monostable whereas in the intermediate region, there are three intersections, one associated with an unstable state and the other two with stable states. <br />
<br />
The bifurcation diagram clearly shows the hysteretic (i.e history-dependent) behaviour of the system in the bistable region. Increasing ν from low to high results in picking the lower branch in the bistable regime, whereas decreasing from high to low takes the system to the upper branch.<br />
<br />
=='''Our Approach'''==<br />
[[Image:The_Y.jpg|thumb|175px|right|Fig. 5. Diagram showing the two ways( indicated by --->) in which the loop is closed.]]<br />
We plan to conduct experiments to explore the multistability of our synthetic genetic circuits by using the approach described by Angeli et al. We use a positive-feedback system, whose open loop can be closed in two different ways <br />
(Fig. 5).<br />
<br />
The transcriptional regulator, LuxR* governs the expression of the promoter, pR. Since the concentration of LuxR* depends on the concentrations of both AI and LuxR, we have a remarkable handle on the feedback. Hence, we look for bistability by varying the concentrations of AI and LuxR and shifting our I/O characteristic curves to intersect the equivalence η=ω(ν=1) line at various points. In contrast, Angeli et al varied the feedback strength (ν) values to explore bistability in their numerical simulations.<br />
<br />
== '''The Parts''' ==<br />
<br />
'''I''' '''''Promoters''''': [[Image:prom_icon.jpeg|40px]] <br />
<br />
*pR [[http://partsregistry.org/Part:BBa_R0062 BBa_R0062]]<br />
*pLac [[http://partsregistry.org/Part:BBa_R0011 BBa_R0011]]<br />
*pTet [[http://partsregistry.org/Part:BBa_R0040 BBa_R0040]]<br />
<br />
'''II''' '''''Regulator''''': [[Image:reg_icon.jpg|40px]]<br />
*LuxR [[http://partsregistry.org/Part:BBa_C0062 BBa_C0062]]<br />
<br />
'''III''' '''''Signalling''''': [[Image:sig_icon.jpg|40px]] <br />
*LuxI --> AI [[http://partsregistry.org/Part:BBa_C0161 BBa_C0161]]<br />
<br />
'''IV''' '''''Inducers''''': [[Image:ind_icon.jpg|40px]] <br />
*IPTG (Isopropyl ß-D-1-thiogalactopyranoside)<br />
*aTc (anhydro tetracycline)<br />
<br />
'''IV''' '''''Reporters''''': [[Image:rep_icon.jpg|40px]] <br />
*CFP (C) [[http://partsregistry.org/Part:BBa_E0020 BBa_E0020]]<br />
*YFP (Y) [[http://partsregistry.org/Part:BBa_E0030 BBa_E0030]]<br />
<br />
== '''The Main Constructs''' ==<br />
[[Image:NET1_closed.jpg|thumb|left|200px|Fig.6 Network 1 by combinatorial construction]] [[Image:NET2_closed.jpg|thumb|right|200px|Fig.7 Network 2 by combinatorial construction]]<br />
<br />
<br />
By combinatorial construction, we have used our network parts to design two positive feedback systems (Figs. 6 & 7). <br />
*pLac luxI.cfp pR luxR.yfp [[http://partsregistry.org/Part:BBa_I726091 BBa_I726091]]<br />
<br />
*pLac luxR.yfp pR luxI.cfp [[http://partsregistry.org/Part:BBa_I726101 BBa_I726101]]<br />
<br />
<br />
The feedback-blocked, open loop system for both the systems can be represented by using the following two constructs as shown in the Fig. 8.<br />
<br />
*pTet luxI.cfp [[http://partsregistry.org/Part:BBa_I726041 BBa_I726041]]<br />
<br />
*pLac luxR.yfp pR cfp [[http://partsregistry.org/Part:BBa_I726081 BBa_I726081]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''Experiments Designed'''==<br />
<br />
[[Image:NET_open.jpg|thumb|200px|right|Fig.8 Diagram illustrating the feedback blocked system of Network 1]]<br />
<br />
==='''The open-loop system for Network 1'''===<br />
<br />
*The feedback step involving production of LuxR from the pR promoter is broken and in its place, LuxR is placed under the regulation of the pLac promoter and the CFP gene is placed under the regulation of the pR promoter.<br />
<br />
*Cells containing the construct pTet luxI.cfp will be initially cultured at a desired aTc concentration (to drive the production of a desired concentration of AI by inducing LuxI expression) at a particular density. The cells will then be separated by centrifugation and the medium containing AI will be added to the growth medium of the cells hosting the construct pLac luxR.yfp pR cfp <br />
<br />
*The fluorescent intensities of the reporter proteins (LuxR.YFP and CFP) will then be obtained using Fluorescence Microscopy and Flow Cytometry, which will now, correspond to a single aTc concentration and cell density.<br />
<br />
*The experiments will be repeated for varying values of aTc concentrations and cell densities to obtain a family of curves relating the CFP and LuxR.YFP as shown (Fig. 9).<br />
<br />
[[Image:LUXRYFPCFP_CURVES.jpg|thumb|200px|left|Fig.9 CFP vs. LuxR.YFP plot]]<br />
<br />
It should be noted that, the O/P (which is fed back as the I/P) in the closed-loop system is different from its counterpart in the open-loop system. In the former, it is LuxR.YFP and in the latter, it is CFP. Hence, an equivalence line (shown in the figure as an inclined dark line), which relates LuxR.YFP and CFP, needs to be obtained. <br />
<br />
The induction of the constructs, pLac luxR.yfp pR cfp and pLac cfp at various concentrations of IPTG will yield us different O/P intensities of CFP and LuxR.YFP for different IPTG concentrations. The expression levels for the two genes (quantified by the fluorescent intensities) at various IPTG concentrations can now be plotted to give the equivalence line. The equivalence line thus generated when superimposed on the family of curves obtained from the open-loop system will give the steady states for the system. Conclusions regarding the stability of the states will be based on the theorem proposed by Angeli et al.<br />
<br />
It has to be noted that, the expression levels of luxI.cfp driven in the open loop will be different from that in the closed loop as they are under two different promoters. Hence, in order to obtain an equivalent concentration of aTc that will give the same expression levels as that of a particular IPTG concentration, we will require an equivalence curve between aTc and IPTG. This will be obtained by inducing the following constructs with varying concentrations of IPTG and aTc.<br />
<br />
1. pLac luxI.cfp [[http://partsregistry.org/Part:BBa_J22231 BBa_I726031]]<br />
<br />
2. pTet luxI.cfp [[http://partsregistry.org/Part:BBa_J22241 BBa_I726041]]<br />
<br />
=='''''The Scaling Argument and its requirement'''''==<br />
<br />
The production of AI is dependent on both LuxI expression and cell density. If the relationship among the three is known, one can achieve various concentrations of AI by sweeping through one of the parameters alone (LuxI or cell density). One can safely assume that at constant LuxI, the amount of AI produced is linearly proportional to cell density and vice-versa. Hence, <br />
<br />
<nowiki> AI = a LuxI * ?</nowiki><br />
<nowiki><br />
<br />
where, a --> constant<br />
? --> cell density</nowiki><br />
<br />
To prove the scaling argument, we find the CFP intensities corresponding to a particular concentration of AI at various aTc concentrations and cell densities. Subsequently, we verify that when the product of cell density and concentration of LuxI is the same in 2 trials, the amount of AI being produced is also the same.<br />
<br />
<br />
=='''Characterisation of Network 2'''==<br />
<br />
The plot for the open loop system in this case can be obtained from the open-loop system of Network 1 by fixing IPTG as the parameter and aTc as the variable input, so that we get the following family of curves (Fig. 10).<br />
[[Image:LUXICFPCFP_CURVES.jpg|thumb|200px|center|Fig.10 Plot of CFP against LuxI.CFP]]<br />
<br />
The steady states can then be obtained as described previously.<br />
<br />
<br />
== '''The Mathematical Model''' ==<br />
The Mathematical model for our system is available [[Media:Model.pdf|here]].<br />
<br />
The following links contain 2 derivations whose results have been used in the model.<br />
*[[Media:derivation_ai.pdf|[AI] Derivation]]<br />
*[[Media:derivation_luxrstar.pdf|[LuxR*] Derivation]]<br />
<br />
=='''References'''==<br />
<br />
1. Christopher M. and Bonnie L. (2005). Quorum Sensing: Cell-to-Cell Communication in Bacteria. '''Annu. Rev. Cell Dev. Biol.''', 21, 319–46.<br />
<br />
2. David Angeli, James E. Ferrell, Jr., and Eduardo D. Sontag (2004). Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems. '''PNAS''', 101, 1822 – 1827.</div>Skunduhttp://2007.igem.org/wiki/index.php/The_CompanyThe Company2007-08-14T09:20:12Z<p>Skundu: </p>
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<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
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{| width="35%"<br />
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<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
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'''The Bangalore iGEM Team, 07'''<br />
<br />
<br />
<br />
<br />
<br />
<br />
|}<br />
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{| width="62%" align="right"<br />
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{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
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! align="center"|[[The Company|The Team]]<br />
! align="center"|[[The Mission|The Mission]]<br />
! align="center"|[[Experiments|Experiments]]<br />
! align="center"|[[e-Notebook|e-Notebook]]<br />
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<br />
<br />
== '''The Company''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
[[Image:Ncbs_Logo.jpg|200px|center]]<br />
<br />
<br />
<br />
<br />
<br />
*Mukund: Chief Mentor, Consultant and The Godfather<br />
*Sugat: Right-hand Man and Manager of Operations<br />
*Nilesh: The Man for all Seasons <br />
*Vivek: Chief Troubleshooter, often his role is confused with one of a Troublemaker <br />
*Krishna: The Sharpshooter <br />
*Navneet: Quorum Sensor(ed) <br />
*Senthil: The Wikiman, also fondly referred to as the Bio Info God<br />
*[[User:Skundu|Shashanka]]: The sili'con' man<br />
*[[User:Smore|Sushant]]: Master Linguist, also called Chow-How Yaake<br />
*Varun: The Voice of Reason and Anti-Reason<br />
*Vini: The Pooh<br />
*''Escherichia coli'' K12Z1: The Workhorse<br />
|<br />
<gallery><br />
Image:Mukund.jpg|Mukund Thattai<br />
Image:Sugat.jpg|Sugat Dhabolkar<br />
Image:Nilesh.jpg|Nilesh Aghera<br />
Image:Vivek.jpg|S. Vivek Raj<br />
<br />
Image:KrishnaR.JPG|Krishna Ramkumar<br />
Image:Navneet.jpg|Navneet Rai<br />
Image:Senthil.jpg|Senthil Kumar<br />
Image:Shashank.jpg|Shashanka S. Kundu<br />
<br />
Image:Sushant.jpg|Sushant More<br />
Image:Varun.jpg|Varun Sreenivasan<br />
Image:Vini.jpg|Vini Gautam<br />
Image:K12Z1.jpg|''E.coli'' K12Z1<br />
</gallery><br />
|}</div>Skunduhttp://2007.igem.org/wiki/index.php/The_CompanyThe Company2007-08-14T09:13:14Z<p>Skundu: </p>
<hr />
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|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
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<br />
<br />
{| align="center"<br />
|-<br />
|'''The Bangalore iGEM Team, 07'''<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
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{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
! align="center"|[[Bangalore|Bangalore]]<br />
! align="center"|[[The Company|The Team]]<br />
! align="center"|[[The Mission|The Mission]]<br />
! align="center"|[[Experiments|Experiments]]<br />
! align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
== '''The Company''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
[[Image:Ncbs_Logo.jpg|200px|center]]<br />
<br />
<br />
<br />
<br />
<br />
*Mukund: Chief Mentor, Consultant and The Godfather<br />
*Sugat: Right-hand Man and Manager of Operations<br />
*Nilesh: The Man for all Seasons <br />
*Vivek: Chief Troubleshooter, often his role is confused with one of a Troublemaker <br />
*Krishna: The Sharpshooter <br />
*Navneet: Quorum Sensor(ed) <br />
*Senthil: The Wikiman, also fondly referred to as the Bio Info God<br />
*[[User:Skundu|Shashanka]]: The sili'con' man<br />
*[[User:Smore|Sushant]]: Master Linguist, also called Chow-How Yaake<br />
*Varun: The Voice of Reason and Anti-Reason<br />
*Vini: The Pooh<br />
*''Escherichia coli'' K12Z1: The Workhorse<br />
|<br />
<gallery><br />
Image:Mukund.jpg|Mukund Thattai<br />
Image:Sugat.jpg|Sugat Dhabolkar<br />
Image:Nilesh.jpg|Nilesh Aghera<br />
Image:Vivek.jpg|S. Vivek Raj<br />
<br />
Image:KrishnaR.JPG|Krishna Ramkumar<br />
Image:Navneet.jpg|Navneet Rai<br />
Image:Senthil.jpg|Senthil Kumar<br />
Image:Shashank.jpg|Shashanka S. Kundu<br />
<br />
Image:Sushant.jpg|Sushant More<br />
Image:Varun.jpg|Varun Sreenivasan<br />
Image:Vini.jpg|Vini Gautam<br />
Image:K12Z1.jpg|''E.coli'' K12Z1<br />
</gallery><br />
|}</div>Skunduhttp://2007.igem.org/wiki/index.php/BangaloreBangalore2007-08-14T09:09:24Z<p>Skundu: </p>
<hr />
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{| width="35%"<br />
|-<br />
|align="center"|<br />
<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|-<br />
|align="justify"|<br />
<font size="2" face="Bookman Old Style">Our team at the National Centre for Biological Sciences, Bangalore comprises undergraduate and graduate students from diverse backgrounds of science. We have gathered together to collectively investigate''' Multistability and Hysteresis in the Combinatorial Synthetic ''Vibrio'' Quorum Sensing Circuits''' designed by us.<br />
<br />
We have also designed a ''Mathematical Model'' for our circuits.</font><br />
|}<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
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<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
!align="center"|[[Bangalore|Bangalore]]<br />
!align="center"|[[The Company|The Team]]<br />
!align="center"|[[The Mission|The Mission]]<br />
!align="center"|[[Experiments|Experiments]]<br />
!align="center"|[[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
== '''Introduction''' ==<br />
<br />
{|<br />
|-<br />
|align="justify"|<br />
<font size="2.5" face="Bookman Old Style"><br />
For a class of feedback systems of arbitrary order, the stability properties and bifurcation diagram of the system can be deduced mathematically from how the system behaves when feedback is blocked (Angeli et al).<br />
<br />
The system is guaranteed to be bistable for some range of feedback strengths provided the feedback-blocked system is monotone and shows a sigmoidal characteristic.<br />
<br />
We have constructed a synthetic genetic circuit similar to the ''Vibrio'' Quorum Sensing System. In our system, the production of the autoinducer is under the control of a gene, the expression of which involves positive feedback. The transcriptional regulator, LuxR* (which is a complex of LuxR and the autoinducer AI) governs the expression of the promoter, pR and we use CFP as the reporter here. Since the concentration of LuxR* depends on the concentrations of both AI and LuxR, we have a remarkable handle on the feedback of this system.<br />
<br />
Note that this is a positive feedback system and its open loop can be closed in two different ways as shown in the following fig.<br />
<br />
<br />
[[Image:The_Y1.jpg|thumb|200px|left| Two ways in which the loop is closed. ]]<br />
<br />
Now we plan to check for the bistability of this circuit.<br />
<br />
Our approach is to conduct a set of open loop experiments on the bacterial strain ''E-coli'' K12Z1. We vary the concentrations of AI and LuxR and get the I/O characteristic curves over a range obtained by varying the concentration of the corresponding inducers (aTc and IPTG). We quantify the expression levels for the genes at different stages by measuring the fluorescence intensities of CFP and YFP reporter proteins in the constructs using Fluorescence Microscopy.<br />
<br />
The sigmoidal family of curves obtained from these open loop experiments when intersect with the superimposed equivalence line will give the steady states for the system. And thus the stability of these states will be concluded thereafter on the theorem proposed by Angeli et al.<br />
<br />
For a detailed description of our project, the experimental design and the mathematical model, go to '''[[The Mission]]'''.<br />
</font><br />
|}<br />
<br />
<br />
== <font size="4">Bangalore iGEM Team 2007</font> ==<br />
<br />
<font size="2" face="Bookman Old Style"><br />
<br />
[[Image:igem_bangalore_07.jpg|thumb|550px|center|<font color="green">'''Top to Bottom''':</font><font color="black"> Navneet, Nilesh, Vivek, Vini, Krishna, Varun, E.coli, Shashanka, Mukund, Senthil and Sushant at the NCBS Amphitheatre.</font>]]<br />
</font></div>Skunduhttp://2007.igem.org/wiki/index.php/BangaloreBangalore2007-08-14T09:05:23Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|120px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{| width="35%"<br />
|-<br />
|align="center"|<br />
<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font><br />
|-<br />
|align="justify"|<br />
<font size="2" face="Bookman Old Style">Our team at the National Centre for Biological Sciences, Bangalore comprises undergraduate and graduate students from diverse backgrounds of science. We have gathered together to collectively investigate''' Multistability and Hysteresis in the Combinatorial Synthetic ''Vibrio'' Quorum Sensing Circuits''' designed by us.<br />
<br />
We have also designed a ''Mathematical Model'' for our circuits.</font><br />
|}<br />
{| width="62%" align="right"<br />
! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
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{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
== '''Introduction''' ==<br />
<br />
{|<br />
|-<br />
|align="justify"|<br />
<font size="2.5" face="Bookman Old Style"><br />
For a class of feedback systems of arbitrary order, the stability properties and bifurcation diagram of the system can be deduced mathematically from how the system behaves when feedback is blocked (Angeli et al).<br />
<br />
The system is guaranteed to be bistable for some range of feedback strengths provided the feedback-blocked system is monotone and shows a sigmoidal characteristic.<br />
<br />
We have constructed a synthetic genetic circuit similar to the ''Vibrio'' Quorum Sensing System. In our system, the production of the autoinducer is under the control of a gene, the expression of which involves positive feedback. The transcriptional regulator, LuxR* (which is a complex of LuxR and the autoinducer AI) governs the expression of the promoter, pR and we use CFP as the reporter here. Since the concentration of LuxR* depends on the concentrations of both AI and LuxR, we have a remarkable handle on the feedback of this system.<br />
<br />
Note that this is a positive feedback system and its open loop can be closed in two different ways as shown in the following fig.<br />
<br />
<br />
[[Image:The_Y1.jpg|thumb|200px|left| Two ways in which the loop is closed. ]]<br />
<br />
Now we plan to check for the bistability of this circuit.<br />
<br />
Our approach is to conduct a set of open loop experiments on the bacterial strain ''E-coli'' K12Z1. We vary the concentrations of AI and LuxR and get the I/O characteristic curves over a range obtained by varying the concentration of the corresponding inducers (aTc and IPTG). We quantify the expression levels for the genes at different stages by measuring the fluorescence intensities of CFP and YFP reporter proteins in the constructs using Fluorescence Microscopy.<br />
<br />
The sigmoidal family of curves obtained from these open loop experiments when intersect with the superimposed equivalence line will give the steady states for the system. And thus the stability of these states will be concluded thereafter on the theorem proposed by Angeli et al.<br />
<br />
For a detailed description of our project, the experimental design and the mathematical model, go to '''[[The Mission]]'''.<br />
</font><br />
|}<br />
<br />
<br />
<br />
== <font size="4">Bangalore iGEM Team 2007</font> ==<br />
<br />
<font size="2" face="Bookman Old Style"><br />
<br />
[[Image:igem_bangalore_07.jpg|thumb|550px|center|<font color="green">'''Top to Bottom''':</font><font color="black"> Navneet, Nilesh, Vivek, Vini, Krishna, Varun, E.coli, Shashanka, Mukund, Senthil and Sushant at the NCBS Amphitheatre.</font>]]<br />
</font></div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-12T07:56:22Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|width="37%"<br />
|- align="center"<br />
|'''The NCBS iGEM 2007 Experiments'''<br />
|-<br />
|align="justify"|<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
*pT.luxI.Cfp<br />
*pL.luxI.Cfp<br />
*pL.luxR.Yfp<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/E-NotebookE-Notebook2007-08-12T07:54:03Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|}<br />
<br />
<br />
{| align="center"<br />
|- <br />
|'''The Bangalore iGEM Journal, 07'''<br />
<br />
|}<br />
<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
<br />
==e-Notebook == <br />
{| align="center" border="0" cellpadding="5" cellspacing="3" <br />
!colspan="7"|June 07 <br />
!colspan="7"| <br />
!colspan="7"|July 07 <br />
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<br />
=='''Protocols''' ==<br />
<br />
'''1) Induction experiments:'''<br />
Grow in LB for 10 hours<br />
Then, induce in Glu M9 for 12 hours at various concns of IPTG/aTc<br />
+ve control: pL YFP in 50,100 uM IPTG <br />
-ve control: K12z1 <br />
Take microscopy images<br />
<br />
'''2) Open loop experiments:'''<br />
i) Morning imaging (followed usually)<br />
Day 0: 0000: Inoculate pTLuxI.C in LB<br />
Day 1: 0100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
1230: Inoculate pLLuxR.Y pRC in LB<br />
2200: Filter required OD flask<br />
2230: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 <br />
Day 2: 1000: Take microscopy images<br />
ii) Night imaging<br />
Day 1: 1100: Inoculate pTLuxI.C in LB<br />
2100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
2330: Inoculate pLLuxR.YpRC in LB<br />
Day 2: 0900: Filter required OD flask<br />
0930: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 in 50 old:50 new 2xM9<br />
2130: Take microscopy images<br />
<br />
==Notes==<br />
*All inductions with IPTG: 0,5,10,50,100,500,1000 uM<br />
<br />
*All inductions with aTc: 0,1,5,10,20,50 ng/mL<br />
<br />
*The open loop experiments to be carried out with following aTc concentrations:<br />
<br />
[A] : 20 ng/ml<br />
<br />
[B] : 10 ng/ml<br />
<br />
[C] : 5 ng/ml<br />
<br />
[D] : 1 ng/ml<br />
<br />
[E] : 50 ng/ml<br />
<br />
[F] : 0 ng/ml<br />
<br />
[AA] : 20 ng/ml (repeat A)<br />
<br />
[FF] : 0 ng/ml (repeat F)<br />
<br />
<br />
The '''''DUPLICATES''''' for each of the above experiments are denoted by [Aa], [Bb], [Cc], [Dd], [Ee] and [Ff] respectively.<br />
<br />
== This Week ==<br />
<br />
{|-<br />
|<br />
Mon, July 2: pL LuxI.C for IPTG-aTc equivalence (morn)<br />
Tue, July 3: pL LuxR.Y for cfp-yfp equivalence, <br />
pT LuxI.C for IPTG-aTc<br />
Wed, July 4: Open loop [E],[F] (aTc) <br />
At OD 0.1: To prove scaling argument for AI.<br />
Thu, July 5: aTC 10,20 ng/mL [X,Y]<br />
DUPLICATES:<br />
---------<br />
At OD 0.2: <br />
Fri, July 6: Open loop: aTC 20,10 ng/mL [A',B'] <br />
Sat, July 7: Open loop: aTC 5,1 ng/mL [C',D']<br />
|}<br />
<br />
<br />
<br />
== Experiments Record ==<br />
*Calibration of the number of colonies obtained at different optical densities.<br />
*Transformation of competent E.coli (strain K12Z1) cells with constructs.<br />
*'''Induction of the construct pLac cfp''' <br />
E.coli cells transformed with the construct pLac cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac cfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
Cells were allowed to grow till the optical density was in the range of 0.05-0.1 (early exponential phase) and were imaged using a phase contrast microscope.<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pLac yfp'''<br />
E.coli cells transformed with the construct pLac yfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac yfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of YFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pTet.luxI.cfp''' <br />
E.coli cells transformed with the construct pTet.luxI.cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of aTc. A stock concentration of 1mg/ml was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!10ug/ml aTc (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pTet luxI cfp(0 ng/ml)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(1 ng/ml)<br />
|0.3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(10 ng/ml)<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(25 ng/ml)<br />
|7.5 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(50 ng/ml)<br />
|15 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(100 ng/ml)<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of aTc.<br />
<br />
*'''Open Loop Trial'''<br />
E. coli cells containing pTet.luxI.cells were first cultured in Luria Bertani medium for 10 hours following which they were transferred to M9 medium where they were induced with aTc (50 ng/ml). The cells were then allowed to grow in M9 for 12 hours.<br />
<br />
The medium (now containing the autoinducer) was filtered and was added to an equal volume of freshly prepared M9 medium containing twice the concentration of glucose. E.coli cells containing pLac.luxR.yfp.pR.cfp (previously grown in LB medium) were inoculated into the M9 medium prepared and were induced with different concentrations of IPTG. Cells were allowed to grow for 12 hours.<br />
<br />
*'''The Scaling Argument'''<br />
<br />
Two sets of experiments would be carried out, where cells would be grown to an optical density of 0.257 & 0.015 respectively in order to check for density dependence of autoinducer production. The supernatent would be filtered out and the one from the OD=0.257 would be scaled down to OD=0.015. The cells would then be imaged using a phase contrast microscope. The scaling argument would be proved if we get the same cfp expression from both.<br />
<br />
== Discussions ==</div>Skunduhttp://2007.igem.org/wiki/index.php/E-NotebookE-Notebook2007-08-12T07:51:16Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|}<br />
<br />
{| align="center"<br />
|-<br />
|'''The Bangalore iGEM Journal, 07'''<br />
<br />
|}<br />
<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
<br />
==e-Notebook == <br />
{| align="center" border="0" cellpadding="5" cellspacing="3" <br />
!colspan="7"|June 07 <br />
!colspan="7"| <br />
!colspan="7"|July 07 <br />
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<br />
=='''Protocols''' ==<br />
<br />
'''1) Induction experiments:'''<br />
Grow in LB for 10 hours<br />
Then, induce in Glu M9 for 12 hours at various concns of IPTG/aTc<br />
+ve control: pL YFP in 50,100 uM IPTG <br />
-ve control: K12z1 <br />
Take microscopy images<br />
<br />
'''2) Open loop experiments:'''<br />
i) Morning imaging (followed usually)<br />
Day 0: 0000: Inoculate pTLuxI.C in LB<br />
Day 1: 0100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
1230: Inoculate pLLuxR.Y pRC in LB<br />
2200: Filter required OD flask<br />
2230: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 <br />
Day 2: 1000: Take microscopy images<br />
ii) Night imaging<br />
Day 1: 1100: Inoculate pTLuxI.C in LB<br />
2100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
2330: Inoculate pLLuxR.YpRC in LB<br />
Day 2: 0900: Filter required OD flask<br />
0930: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 in 50 old:50 new 2xM9<br />
2130: Take microscopy images<br />
<br />
==Notes==<br />
*All inductions with IPTG: 0,5,10,50,100,500,1000 uM<br />
<br />
*All inductions with aTc: 0,1,5,10,20,50 ng/mL<br />
<br />
*The open loop experiments to be carried out with following aTc concentrations:<br />
<br />
[A] : 20 ng/ml<br />
<br />
[B] : 10 ng/ml<br />
<br />
[C] : 5 ng/ml<br />
<br />
[D] : 1 ng/ml<br />
<br />
[E] : 50 ng/ml<br />
<br />
[F] : 0 ng/ml<br />
<br />
[AA] : 20 ng/ml (repeat A)<br />
<br />
[FF] : 0 ng/ml (repeat F)<br />
<br />
<br />
The '''''DUPLICATES''''' for each of the above experiments are denoted by [Aa], [Bb], [Cc], [Dd], [Ee] and [Ff] respectively.<br />
<br />
== This Week ==<br />
<br />
{|-<br />
|<br />
Mon, July 2: pL LuxI.C for IPTG-aTc equivalence (morn)<br />
Tue, July 3: pL LuxR.Y for cfp-yfp equivalence, <br />
pT LuxI.C for IPTG-aTc<br />
Wed, July 4: Open loop [E],[F] (aTc) <br />
At OD 0.1: To prove scaling argument for AI.<br />
Thu, July 5: aTC 10,20 ng/mL [X,Y]<br />
DUPLICATES:<br />
---------<br />
At OD 0.2: <br />
Fri, July 6: Open loop: aTC 20,10 ng/mL [A',B'] <br />
Sat, July 7: Open loop: aTC 5,1 ng/mL [C',D']<br />
|}<br />
<br />
<br />
<br />
== Experiments Record ==<br />
*Calibration of the number of colonies obtained at different optical densities.<br />
*Transformation of competent E.coli (strain K12Z1) cells with constructs.<br />
*'''Induction of the construct pLac cfp''' <br />
E.coli cells transformed with the construct pLac cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac cfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
Cells were allowed to grow till the optical density was in the range of 0.05-0.1 (early exponential phase) and were imaged using a phase contrast microscope.<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pLac yfp'''<br />
E.coli cells transformed with the construct pLac yfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac yfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of YFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pTet.luxI.cfp''' <br />
E.coli cells transformed with the construct pTet.luxI.cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of aTc. A stock concentration of 1mg/ml was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!10ug/ml aTc (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pTet luxI cfp(0 ng/ml)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(1 ng/ml)<br />
|0.3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(10 ng/ml)<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(25 ng/ml)<br />
|7.5 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(50 ng/ml)<br />
|15 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(100 ng/ml)<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of aTc.<br />
<br />
*'''Open Loop Trial'''<br />
E. coli cells containing pTet.luxI.cells were first cultured in Luria Bertani medium for 10 hours following which they were transferred to M9 medium where they were induced with aTc (50 ng/ml). The cells were then allowed to grow in M9 for 12 hours.<br />
<br />
The medium (now containing the autoinducer) was filtered and was added to an equal volume of freshly prepared M9 medium containing twice the concentration of glucose. E.coli cells containing pLac.luxR.yfp.pR.cfp (previously grown in LB medium) were inoculated into the M9 medium prepared and were induced with different concentrations of IPTG. Cells were allowed to grow for 12 hours.<br />
<br />
*'''The Scaling Argument'''<br />
<br />
Two sets of experiments would be carried out, where cells would be grown to an optical density of 0.257 & 0.015 respectively in order to check for density dependence of autoinducer production. The supernatent would be filtered out and the one from the OD=0.257 would be scaled down to OD=0.015. The cells would then be imaged using a phase contrast microscope. The scaling argument would be proved if we get the same cfp expression from both.<br />
<br />
== Discussions ==</div>Skunduhttp://2007.igem.org/wiki/index.php/E-NotebookE-Notebook2007-08-12T07:50:55Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|}<br />
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{| align="center"<br />
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|'''The Bangalore iGEM Journal, 07'''<br />
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![[e-Notebook|e-Notebook]]<br />
|}<br />
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<br />
<br />
==e-Notebook == <br />
{| align="center" border="0" cellpadding="5" cellspacing="3" <br />
!colspan="7"|June 07 <br />
!colspan="7"| <br />
!colspan="7"|July 07 <br />
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<br />
=='''Protocols''' ==<br />
<br />
'''1) Induction experiments:'''<br />
Grow in LB for 10 hours<br />
Then, induce in Glu M9 for 12 hours at various concns of IPTG/aTc<br />
+ve control: pL YFP in 50,100 uM IPTG <br />
-ve control: K12z1 <br />
Take microscopy images<br />
<br />
'''2) Open loop experiments:'''<br />
i) Morning imaging (followed usually)<br />
Day 0: 0000: Inoculate pTLuxI.C in LB<br />
Day 1: 0100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
1230: Inoculate pLLuxR.Y pRC in LB<br />
2200: Filter required OD flask<br />
2230: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 <br />
Day 2: 1000: Take microscopy images<br />
ii) Night imaging<br />
Day 1: 1100: Inoculate pTLuxI.C in LB<br />
2100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
2330: Inoculate pLLuxR.YpRC in LB<br />
Day 2: 0900: Filter required OD flask<br />
0930: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 in 50 old:50 new 2xM9<br />
2130: Take microscopy images<br />
<br />
==Notes==<br />
*All inductions with IPTG: 0,5,10,50,100,500,1000 uM<br />
<br />
*All inductions with aTc: 0,1,5,10,20,50 ng/mL<br />
<br />
*The open loop experiments to be carried out with following aTc concentrations:<br />
<br />
[A] : 20 ng/ml<br />
<br />
[B] : 10 ng/ml<br />
<br />
[C] : 5 ng/ml<br />
<br />
[D] : 1 ng/ml<br />
<br />
[E] : 50 ng/ml<br />
<br />
[F] : 0 ng/ml<br />
<br />
[AA] : 20 ng/ml (repeat A)<br />
<br />
[FF] : 0 ng/ml (repeat F)<br />
<br />
<br />
The '''''DUPLICATES''''' for each of the above experiments are denoted by [Aa], [Bb], [Cc], [Dd], [Ee] and [Ff] respectively.<br />
<br />
== This Week ==<br />
<br />
{|-<br />
|<br />
Mon, July 2: pL LuxI.C for IPTG-aTc equivalence (morn)<br />
Tue, July 3: pL LuxR.Y for cfp-yfp equivalence, <br />
pT LuxI.C for IPTG-aTc<br />
Wed, July 4: Open loop [E],[F] (aTc) <br />
At OD 0.1: To prove scaling argument for AI.<br />
Thu, July 5: aTC 10,20 ng/mL [X,Y]<br />
DUPLICATES:<br />
---------<br />
At OD 0.2: <br />
Fri, July 6: Open loop: aTC 20,10 ng/mL [A',B'] <br />
Sat, July 7: Open loop: aTC 5,1 ng/mL [C',D']<br />
|}<br />
<br />
<br />
<br />
== Experiments Record ==<br />
*Calibration of the number of colonies obtained at different optical densities.<br />
*Transformation of competent E.coli (strain K12Z1) cells with constructs.<br />
*'''Induction of the construct pLac cfp''' <br />
E.coli cells transformed with the construct pLac cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac cfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
Cells were allowed to grow till the optical density was in the range of 0.05-0.1 (early exponential phase) and were imaged using a phase contrast microscope.<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pLac yfp'''<br />
E.coli cells transformed with the construct pLac yfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac yfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of YFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pTet.luxI.cfp''' <br />
E.coli cells transformed with the construct pTet.luxI.cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of aTc. A stock concentration of 1mg/ml was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!10ug/ml aTc (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pTet luxI cfp(0 ng/ml)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(1 ng/ml)<br />
|0.3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(10 ng/ml)<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(25 ng/ml)<br />
|7.5 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(50 ng/ml)<br />
|15 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(100 ng/ml)<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of aTc.<br />
<br />
*'''Open Loop Trial'''<br />
E. coli cells containing pTet.luxI.cells were first cultured in Luria Bertani medium for 10 hours following which they were transferred to M9 medium where they were induced with aTc (50 ng/ml). The cells were then allowed to grow in M9 for 12 hours.<br />
<br />
The medium (now containing the autoinducer) was filtered and was added to an equal volume of freshly prepared M9 medium containing twice the concentration of glucose. E.coli cells containing pLac.luxR.yfp.pR.cfp (previously grown in LB medium) were inoculated into the M9 medium prepared and were induced with different concentrations of IPTG. Cells were allowed to grow for 12 hours.<br />
<br />
*'''The Scaling Argument'''<br />
<br />
Two sets of experiments would be carried out, where cells would be grown to an optical density of 0.257 & 0.015 respectively in order to check for density dependence of autoinducer production. The supernatent would be filtered out and the one from the OD=0.257 would be scaled down to OD=0.015. The cells would then be imaged using a phase contrast microscope. The scaling argument would be proved if we get the same cfp expression from both.<br />
<br />
== Discussions ==</div>Skunduhttp://2007.igem.org/wiki/index.php/E-NotebookE-Notebook2007-08-12T07:50:34Z<p>Skundu: </p>
<hr />
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|'''The Bangalore iGEM Journal, 07'''<br />
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==e-Notebook == <br />
{| align="center" border="0" cellpadding="5" cellspacing="3" <br />
!colspan="7"|June 07 <br />
!colspan="7"| <br />
!colspan="7"|July 07 <br />
|-<br />
|'''S''' <br />
|M <br />
|T <br />
|W <br />
|T <br />
|F <br />
|S <br />
| <br />
| <br />
| <br />
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| <br />
| <br />
| <br />
|'''S''' <br />
|M <br />
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|T <br />
|F <br />
|S <br />
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|[[June 1|1]] <br />
|2 <br />
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| <br />
|[[July 1|1]] <br />
|[[July 2|2]] <br />
|[[July 3|3]] <br />
|[[July 4|4]] <br />
|[[July 5|5]] <br />
|[[July 6|6]] <br />
|[[July 7|7]] <br />
|-<br />
|3 <br />
|[[June 4|4]] <br />
|[[June 5|5]] <br />
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|[[June 8|8]] <br />
|9 <br />
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|[[July 8|8]] <br />
|[[July 9|9]] <br />
|[[July 10|10]] <br />
|[[July 11|11]] <br />
|[[July 12|12]] <br />
|[[July 13|13]] <br />
|[[July 14|14]] <br />
|-<br />
|10 <br />
|[[June 11|11]] <br />
|[[June 12|12]] <br />
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|[[June 14|14]] <br />
|[[June 15|15]] <br />
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|[[June 20|20]] <br />
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|[[June 22|22]] <br />
|[[June 23|23]] <br />
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|[[June 24|24]] <br />
|[[June 25|25]] <br />
|[[June 26|26]] <br />
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|[[June 28|28]] <br />
|[[June 29|29]] <br />
|[[June 30|30]] <br />
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|29 <br />
|30 <br />
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|}<br />
<br />
=='''Protocols''' ==<br />
<br />
'''1) Induction experiments:'''<br />
Grow in LB for 10 hours<br />
Then, induce in Glu M9 for 12 hours at various concns of IPTG/aTc<br />
+ve control: pL YFP in 50,100 uM IPTG <br />
-ve control: K12z1 <br />
Take microscopy images<br />
<br />
'''2) Open loop experiments:'''<br />
i) Morning imaging (followed usually)<br />
Day 0: 0000: Inoculate pTLuxI.C in LB<br />
Day 1: 0100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
1230: Inoculate pLLuxR.Y pRC in LB<br />
2200: Filter required OD flask<br />
2230: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 <br />
Day 2: 1000: Take microscopy images<br />
ii) Night imaging<br />
Day 1: 1100: Inoculate pTLuxI.C in LB<br />
2100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
2330: Inoculate pLLuxR.YpRC in LB<br />
Day 2: 0900: Filter required OD flask<br />
0930: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 in 50 old:50 new 2xM9<br />
2130: Take microscopy images<br />
<br />
==Notes==<br />
*All inductions with IPTG: 0,5,10,50,100,500,1000 uM<br />
<br />
*All inductions with aTc: 0,1,5,10,20,50 ng/mL<br />
<br />
*The open loop experiments to be carried out with following aTc concentrations:<br />
<br />
[A] : 20 ng/ml<br />
<br />
[B] : 10 ng/ml<br />
<br />
[C] : 5 ng/ml<br />
<br />
[D] : 1 ng/ml<br />
<br />
[E] : 50 ng/ml<br />
<br />
[F] : 0 ng/ml<br />
<br />
[AA] : 20 ng/ml (repeat A)<br />
<br />
[FF] : 0 ng/ml (repeat F)<br />
<br />
<br />
The '''''DUPLICATES''''' for each of the above experiments are denoted by [Aa], [Bb], [Cc], [Dd], [Ee] and [Ff] respectively.<br />
<br />
== This Week ==<br />
<br />
{|-<br />
|<br />
Mon, July 2: pL LuxI.C for IPTG-aTc equivalence (morn)<br />
Tue, July 3: pL LuxR.Y for cfp-yfp equivalence, <br />
pT LuxI.C for IPTG-aTc<br />
Wed, July 4: Open loop [E],[F] (aTc) <br />
At OD 0.1: To prove scaling argument for AI.<br />
Thu, July 5: aTC 10,20 ng/mL [X,Y]<br />
DUPLICATES:<br />
---------<br />
At OD 0.2: <br />
Fri, July 6: Open loop: aTC 20,10 ng/mL [A',B'] <br />
Sat, July 7: Open loop: aTC 5,1 ng/mL [C',D']<br />
|}<br />
<br />
<br />
<br />
== Experiments Record ==<br />
*Calibration of the number of colonies obtained at different optical densities.<br />
*Transformation of competent E.coli (strain K12Z1) cells with constructs.<br />
*'''Induction of the construct pLac cfp''' <br />
E.coli cells transformed with the construct pLac cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac cfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
Cells were allowed to grow till the optical density was in the range of 0.05-0.1 (early exponential phase) and were imaged using a phase contrast microscope.<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pLac yfp'''<br />
E.coli cells transformed with the construct pLac yfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac yfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of YFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pTet.luxI.cfp''' <br />
E.coli cells transformed with the construct pTet.luxI.cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of aTc. A stock concentration of 1mg/ml was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!10ug/ml aTc (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pTet luxI cfp(0 ng/ml)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(1 ng/ml)<br />
|0.3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(10 ng/ml)<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(25 ng/ml)<br />
|7.5 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(50 ng/ml)<br />
|15 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(100 ng/ml)<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of aTc.<br />
<br />
*'''Open Loop Trial'''<br />
E. coli cells containing pTet.luxI.cells were first cultured in Luria Bertani medium for 10 hours following which they were transferred to M9 medium where they were induced with aTc (50 ng/ml). The cells were then allowed to grow in M9 for 12 hours.<br />
<br />
The medium (now containing the autoinducer) was filtered and was added to an equal volume of freshly prepared M9 medium containing twice the concentration of glucose. E.coli cells containing pLac.luxR.yfp.pR.cfp (previously grown in LB medium) were inoculated into the M9 medium prepared and were induced with different concentrations of IPTG. Cells were allowed to grow for 12 hours.<br />
<br />
*'''The Scaling Argument'''<br />
<br />
Two sets of experiments would be carried out, where cells would be grown to an optical density of 0.257 & 0.015 respectively in order to check for density dependence of autoinducer production. The supernatent would be filtered out and the one from the OD=0.257 would be scaled down to OD=0.015. The cells would then be imaged using a phase contrast microscope. The scaling argument would be proved if we get the same cfp expression from both.<br />
<br />
== Discussions ==</div>Skunduhttp://2007.igem.org/wiki/index.php/E-NotebookE-Notebook2007-08-12T07:50:11Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|}<br />
<br />
<br />
{| align="center"<br />
|-<br />
|'''The Bangalore iGEM Team, 07'''<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
|}<br />
<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
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<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
<br />
==e-Notebook == <br />
{| align="center" border="0" cellpadding="5" cellspacing="3" <br />
!colspan="7"|June 07 <br />
!colspan="7"| <br />
!colspan="7"|July 07 <br />
|-<br />
|'''S''' <br />
|M <br />
|T <br />
|W <br />
|T <br />
|F <br />
|S <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|'''S''' <br />
|M <br />
|T <br />
|W <br />
|T <br />
|F <br />
|S <br />
|- <br />
|<br />
|<br />
|<br />
|<br />
|<br />
|[[June 1|1]] <br />
|2 <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|[[July 1|1]] <br />
|[[July 2|2]] <br />
|[[July 3|3]] <br />
|[[July 4|4]] <br />
|[[July 5|5]] <br />
|[[July 6|6]] <br />
|[[July 7|7]] <br />
|-<br />
|3 <br />
|[[June 4|4]] <br />
|[[June 5|5]] <br />
|[[June 6|6]] <br />
|[[June 7|7]] <br />
|[[June 8|8]] <br />
|9 <br />
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|[[July 8|8]] <br />
|[[July 9|9]] <br />
|[[July 10|10]] <br />
|[[July 11|11]] <br />
|[[July 12|12]] <br />
|[[July 13|13]] <br />
|[[July 14|14]] <br />
|-<br />
|10 <br />
|[[June 11|11]] <br />
|[[June 12|12]] <br />
|[[June 13|13]] <br />
|[[June 14|14]] <br />
|[[June 15|15]] <br />
|[[June 16|16]] <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|[[July 15|15]] <br />
|16 <br />
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|20 <br />
|21 <br />
|- <br />
|[[June 17|17]] <br />
|[[June 18|18]] <br />
|[[June 19|19]] <br />
|[[June 20|20]] <br />
|[[June 21|21]] <br />
|[[June 22|22]] <br />
|[[June 23|23]] <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|22 <br />
|23 <br />
|24 <br />
|25 <br />
|26 <br />
|27 <br />
|28 <br />
|- <br />
|[[June 24|24]] <br />
|[[June 25|25]] <br />
|[[June 26|26]] <br />
|[[June 27|27]] <br />
|[[June 28|28]] <br />
|[[June 29|29]] <br />
|[[June 30|30]] <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|29 <br />
|30 <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|}<br />
<br />
=='''Protocols''' ==<br />
<br />
'''1) Induction experiments:'''<br />
Grow in LB for 10 hours<br />
Then, induce in Glu M9 for 12 hours at various concns of IPTG/aTc<br />
+ve control: pL YFP in 50,100 uM IPTG <br />
-ve control: K12z1 <br />
Take microscopy images<br />
<br />
'''2) Open loop experiments:'''<br />
i) Morning imaging (followed usually)<br />
Day 0: 0000: Inoculate pTLuxI.C in LB<br />
Day 1: 0100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
1230: Inoculate pLLuxR.Y pRC in LB<br />
2200: Filter required OD flask<br />
2230: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 <br />
Day 2: 1000: Take microscopy images<br />
ii) Night imaging<br />
Day 1: 1100: Inoculate pTLuxI.C in LB<br />
2100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]<br />
2330: Inoculate pLLuxR.YpRC in LB<br />
Day 2: 0900: Filter required OD flask<br />
0930: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9<br />
-ve control: K12z1 in 50 old:50 new 2xM9<br />
2130: Take microscopy images<br />
<br />
==Notes==<br />
*All inductions with IPTG: 0,5,10,50,100,500,1000 uM<br />
<br />
*All inductions with aTc: 0,1,5,10,20,50 ng/mL<br />
<br />
*The open loop experiments to be carried out with following aTc concentrations:<br />
<br />
[A] : 20 ng/ml<br />
<br />
[B] : 10 ng/ml<br />
<br />
[C] : 5 ng/ml<br />
<br />
[D] : 1 ng/ml<br />
<br />
[E] : 50 ng/ml<br />
<br />
[F] : 0 ng/ml<br />
<br />
[AA] : 20 ng/ml (repeat A)<br />
<br />
[FF] : 0 ng/ml (repeat F)<br />
<br />
<br />
The '''''DUPLICATES''''' for each of the above experiments are denoted by [Aa], [Bb], [Cc], [Dd], [Ee] and [Ff] respectively.<br />
<br />
== This Week ==<br />
<br />
{|-<br />
|<br />
Mon, July 2: pL LuxI.C for IPTG-aTc equivalence (morn)<br />
Tue, July 3: pL LuxR.Y for cfp-yfp equivalence, <br />
pT LuxI.C for IPTG-aTc<br />
Wed, July 4: Open loop [E],[F] (aTc) <br />
At OD 0.1: To prove scaling argument for AI.<br />
Thu, July 5: aTC 10,20 ng/mL [X,Y]<br />
DUPLICATES:<br />
---------<br />
At OD 0.2: <br />
Fri, July 6: Open loop: aTC 20,10 ng/mL [A',B'] <br />
Sat, July 7: Open loop: aTC 5,1 ng/mL [C',D']<br />
|}<br />
<br />
<br />
<br />
== Experiments Record ==<br />
*Calibration of the number of colonies obtained at different optical densities.<br />
*Transformation of competent E.coli (strain K12Z1) cells with constructs.<br />
*'''Induction of the construct pLac cfp''' <br />
E.coli cells transformed with the construct pLac cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac cfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac cfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
Cells were allowed to grow till the optical density was in the range of 0.05-0.1 (early exponential phase) and were imaged using a phase contrast microscope.<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pLac yfp'''<br />
E.coli cells transformed with the construct pLac yfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of IPTG. A stock concentration of 100mM was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!100mM IPTG (ul)<br />
!1mM IPTG (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pLac yfp (0 uM)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1 uM)<br />
| -<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (10 uM)<br />
| -<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (50 uM)<br />
|1.5 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (100 uM)<br />
|3 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pLac yfp (1000 uM)<br />
|30 ul<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of YFP was seen to increase with increasing concentrations of the IPTG.<br />
<br />
*'''Induction of the construct pTet.luxI.cfp''' <br />
E.coli cells transformed with the construct pTet.luxI.cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.<br />
<br />
The cells were then transferred into a minimal medium (M9 medium) and were induced with the following concentrations of aTc. A stock concentration of 1mg/ml was used.<br />
{| cellpadding="2" cellspacing="3" border="1"<br />
!Sample<br />
!10ug/ml aTc (ul)<br />
!Inoculum volume from LB (ul)<br />
!Volume of M9 added (ml)<br />
|-align="center"<br />
|pTet luxI cfp(0 ng/ml)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(1 ng/ml)<br />
|0.3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(10 ng/ml)<br />
|3 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(25 ng/ml)<br />
|7.5 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(50 ng/ml)<br />
|15 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|pTet luxI cfp(100 ng/ml)<br />
|30 ul<br />
|3 ul<br />
|3 ml<br />
|-align="center"<br />
|K12Z1 (Autoflourescence)<br />
| -<br />
|3 ul<br />
|3 ml<br />
|}<br />
<br />
'''Results:'''<br />
The mean fluorescence of CFP was seen to increase with increasing concentrations of aTc.<br />
<br />
*'''Open Loop Trial'''<br />
E. coli cells containing pTet.luxI.cells were first cultured in Luria Bertani medium for 10 hours following which they were transferred to M9 medium where they were induced with aTc (50 ng/ml). The cells were then allowed to grow in M9 for 12 hours.<br />
<br />
The medium (now containing the autoinducer) was filtered and was added to an equal volume of freshly prepared M9 medium containing twice the concentration of glucose. E.coli cells containing pLac.luxR.yfp.pR.cfp (previously grown in LB medium) were inoculated into the M9 medium prepared and were induced with different concentrations of IPTG. Cells were allowed to grow for 12 hours.<br />
<br />
*'''The Scaling Argument'''<br />
<br />
Two sets of experiments would be carried out, where cells would be grown to an optical density of 0.257 & 0.015 respectively in order to check for density dependence of autoinducer production. The supernatent would be filtered out and the one from the OD=0.257 would be scaled down to OD=0.015. The cells would then be imaged using a phase contrast microscope. The scaling argument would be proved if we get the same cfp expression from both.<br />
<br />
== Discussions ==</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-12T07:49:39Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|width="37%"<br />
|- align="center"<br />
|'''The NCBS iGEM 2007 Experiments'''<br />
|- align="justify"<br />
|<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
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![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
*pT.luxI.Cfp<br />
*pL.luxI.Cfp<br />
*pL.luxR.Yfp<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/The_MissionThe Mission2007-08-12T07:45:52Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|}<br />
<br />
<br />
{| align="center"<br />
|-<br />
|'''The Bangalore iGEM Project Plan, 07'''<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
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<br />
<br />
== '''The Objective''' ==<br />
<br />
To investigate multistability and hysteresis in combinatorially constructed synthetic ''Vibrio'' quorum sensing circuits<br />
<br />
== '''The Target - ''Vibrio'' Quorum Sensing System''' ==<br />
<br />
[[Image:vibrio_qs.jpg|thumb|200px|Fig. 1: Quorum sensing in Vibrio fischeri - a LuxI-R signalling circuit. Red triangles indicate the autoinducer that is produced by LuxI. OM, outer membrane; IM, inner membrane.]]<br />
<br />
Quorum sensing is a phenomenon by which bacteria sense a critical cell density before turning on the expression of certain genes. It involves the gradual build-up of a chemical termed the 'autoinducer' in the cell. The autoinducer freely diffuses across the cell membrane and hence, its concentration is population density dependent. When the concentration crosses a threshold, the bacteria switch to a different physiological state such as bioluminescence, virulent gene expression, and bio-film formation. <br />
<br />
In ''Vibrio fischeri'', when the population density (and hence the concentration of autoinducer) crosses a certain threshold, the expression of a set of genes that is required for bioluminescence is turned on. The production of the autoinducer is under the control of a gene, the expression of which involves ''positive feedback''. Figure 1 shows the various components of this system (Ref. 1).<br />
<br />
View a powerpoint presentation. [[Media:QSppt.ppt|Click here.]]<br />
<br />
==='''Why did we pick this system?'''===<br />
<br />
The Vibrio quorum sensing system involves a well-defined set of genes and a promoter, and has a degree of complexity that offers wide scope for exploration. Additionally, the concentration of the active transcriptional regulator, LuxR* is dependent on 3 factors:<br />
i) The concentration of LuxI <br />
ii) The population density <br />
iii) The concentration of LuxR<br />
<br />
All these factors can be experimentally controlled. Thus, the nature of the system offers one an extremely good handle on the feedback strength of the genetic circuit.<br />
<br />
=='''A note on Multistability and Hysteresis'''==<br />
[[Image:Break_feedback_loop.jpg|thumb|200px|left|Fig. 2: Schematic view of a feedback system before (Left) and after (Right) breaking the feedback loop. ‘ω’ is the input of the open-loop system and ‘η’ is the output.]]<br />
<br />
In the recent past, multistability has been an important recurring theme in studies on cell signalling. Angeli et al (Ref. 2) have shown that for a class of feedback systems of arbitrary order, the stability properties and bifurcation diagram of the system can be deduced mathematically from how the system behaves when feedback is blocked. [[Image:Io_curve.jpg|thumb|200px|right|Fig. 3: Steady state I/O static characteristic curve for the open loop of the mutually inhibitory Cdc2-Cyclin B/Wee1 feedback system (red). The solid blue line represents η as a function of ω for unitary feedback. ]]The system is guaranteed to be bistable for some range of feedback strengths provided the feedback-blocked system is monotone and shows a sigmoidal characteristic. <br />
<br />
A simple graphical method can be used to deduce the stability behaviour of such systems (Ref. 2). The key feature of this approach is to view the positive feedback system as a feedback closure of its corresponding 'open loop' system. This open loop system is obtained by breaking the feedback loop at the point of feedback (Fig. 2).<br />
<br />
Now, one can experimentally manipulate the amount of input (ω) and monitor the output (η) as a function of ω. The fixed points of the corresponding closed loop system are then obtained by intersecting η=f(ω) with the straight line, η=(1/ν)ω, where ' ν ' is the feedback strength. At these points of intersection, the open loop system exactly mimics the closed loop system. As shown in Fig. 3, they represent two stable steady states (I and III) and one unstable steady state (II).<br />
<br />
[[Image:Bifurcation.jpg|thumb|185px|left|Fig. 4: Bifurcation diagram, showing bistability when the feedback strength ‘ν’ is between ~0.83 and ~1.8. ]]<br />
<br />
All positive-feedback, multistable systems show the associated property of ''hysteresis''. This can be explained by analysing the corresponding bifurcation diagram, which is a plot of the steady states as a function of the feedback strength (see Fig. 4). To do this, one can vary the feedback strength, ‘ν’ and find the different points at which the I/O characteristic curve intersects the equivalence line η=(1/ν)ω. At high and low feedback strength values, the system is monostable whereas in the intermediate region, there are three intersections, one associated with an unstable state and the other two with stable states. <br />
<br />
The bifurcation diagram clearly shows the hysteretic (i.e history-dependent) behaviour of the system in the bistable region. Increasing ν from low to high results in picking the lower branch in the bistable regime, whereas decreasing from high to low takes the system to the upper branch.<br />
<br />
=='''Our Approach'''==<br />
[[Image:The_Y.jpg|thumb|175px|right|Fig. 5. Diagram showing the two ways( indicated by --->) in which the loop is closed.]]<br />
We plan to conduct experiments to explore the multistability of our synthetic genetic circuits by using the approach described by Angeli et al. We use a positive-feedback system, whose open loop can be closed in two different ways <br />
(Fig. 5).<br />
<br />
The transcriptional regulator, LuxR* governs the expression of the promoter, pR. Since the concentration of LuxR* depends on the concentrations of both AI and LuxR, we have a remarkable handle on the feedback. Hence, we look for bistability by varying the concentrations of AI and LuxR and shifting our I/O characteristic curves to intersect the equivalence η=ω(ν=1) line at various points. In contrast, Angeli et al varied the feedback strength (ν) values to explore bistability in their numerical simulations.<br />
<br />
== '''The Parts''' ==<br />
<br />
'''I''' '''''Promoters''''': [[Image:prom_icon.jpeg|40px]] <br />
<br />
*pR [[http://partsregistry.org/Part:BBa_R0062 BBa_R0062]]<br />
*pLac [[http://partsregistry.org/Part:BBa_R0011 BBa_R0011]]<br />
*pTet [[http://partsregistry.org/Part:BBa_R0040 BBa_R0040]]<br />
<br />
'''II''' '''''Regulator''''': [[Image:reg_icon.jpg|40px]]<br />
*LuxR [[http://partsregistry.org/Part:BBa_C0062 BBa_C0062]]<br />
<br />
'''III''' '''''Signalling''''': [[Image:sig_icon.jpg|40px]] <br />
*LuxI --> AI [[http://partsregistry.org/Part:BBa_C0161 BBa_C0161]]<br />
<br />
'''IV''' '''''Inducers''''': [[Image:ind_icon.jpg|40px]] <br />
*IPTG (Isopropyl ß-D-1-thiogalactopyranoside)<br />
*aTc (anhydro tetracycline)<br />
<br />
'''IV''' '''''Reporters''''': [[Image:rep_icon.jpg|40px]] <br />
*CFP (C) [[http://partsregistry.org/Part:BBa_E0020 BBa_E0020]]<br />
*YFP (Y) [[http://partsregistry.org/Part:BBa_E0030 BBa_E0030]]<br />
<br />
== '''The Main Constructs''' ==<br />
[[Image:NET1_closed.jpg|thumb|left|200px|Fig.6 Network 1 by combinatorial construction]] [[Image:NET2_closed.jpg|thumb|right|200px|Fig.7 Network 2 by combinatorial construction]]<br />
<br />
<br />
By combinatorial construction, we have used our network parts to design two positive feedback systems (Figs. 6 & 7). <br />
*pLac luxI.cfp pR luxR.yfp [[http://partsregistry.org/Part:BBa_I726091 BBa_I726091]]<br />
<br />
*pLac luxR.yfp pR luxI.cfp [[http://partsregistry.org/Part:BBa_I726101 BBa_I726101]]<br />
<br />
<br />
The feedback-blocked, open loop system for both the systems can be represented by using the following two constructs as shown in the Fig. 8.<br />
<br />
*pTet luxI.cfp [[http://partsregistry.org/Part:BBa_I726041 BBa_I726041]]<br />
<br />
*pLac luxR.yfp pR cfp [[http://partsregistry.org/Part:BBa_I726081 BBa_I726081]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''Experiments Designed'''==<br />
<br />
[[Image:NET_open.jpg|thumb|200px|right|Fig.8 Diagram illustrating the feedback blocked system of Network 1]]<br />
<br />
==='''The open-loop system for Network 1'''===<br />
<br />
*The feedback step involving production of LuxR from the pR promoter is broken and in its place, LuxR is placed under the regulation of the pLac promoter and the CFP gene is placed under the regulation of the pR promoter.<br />
<br />
*Cells containing the construct pTet luxI.cfp will be initially cultured at a desired aTc concentration (to drive the production of a desired concentration of AI by inducing LuxI expression) at a particular density. The cells will then be separated by centrifugation and the medium containing AI will be added to the growth medium of the cells hosting the construct pLac luxR.yfp pR cfp <br />
<br />
*The fluorescent intensities of the reporter proteins (LuxR.YFP and CFP) will then be obtained using Fluorescence Microscopy and Flow Cytometry, which will now, correspond to a single aTc concentration and cell density.<br />
<br />
*The experiments will be repeated for varying values of aTc concentrations and cell densities to obtain a family of curves relating the CFP and LuxR.YFP as shown (Fig. 9).<br />
<br />
[[Image:LUXRYFPCFP_CURVES.jpg|thumb|200px|left|Fig.9 CFP vs. LuxR.YFP plot]]<br />
<br />
It should be noted that, the O/P (which is fed back as the I/P) in the closed-loop system is different from its counterpart in the open-loop system. In the former, it is LuxR.YFP and in the latter, it is CFP. Hence, an equivalence line (shown in the figure as an inclined dark line), which relates LuxR.YFP and CFP, needs to be obtained. <br />
<br />
The induction of the constructs, pLac luxR.yfp pR cfp and pLac cfp at various concentrations of IPTG will yield us different O/P intensities of CFP and LuxR.YFP for different IPTG concentrations. The expression levels for the two genes (quantified by the fluorescent intensities) at various IPTG concentrations can now be plotted to give the equivalence line. The equivalence line thus generated when superimposed on the family of curves obtained from the open-loop system will give the steady states for the system. Conclusions regarding the stability of the states will be based on the theorem proposed by Angeli et al.<br />
<br />
It has to be noted that, the expression levels of luxI.cfp driven in the open loop will be different from that in the closed loop as they are under two different promoters. Hence, in order to obtain an equivalent concentration of aTc that will give the same expression levels as that of a particular IPTG concentration, we will require an equivalence curve between aTc and IPTG. This will be obtained by inducing the following constructs with varying concentrations of IPTG and aTc.<br />
<br />
1. pLac luxI.cfp [[http://partsregistry.org/Part:BBa_J22231 BBa_I726031]]<br />
<br />
2. pTet luxI.cfp [[http://partsregistry.org/Part:BBa_J22241 BBa_I726041]]<br />
<br />
=='''''The Scaling Argument and its requirement'''''==<br />
<br />
The production of AI is dependent on both LuxI expression and cell density. If the relationship among the three is known, one can achieve various concentrations of AI by sweeping through one of the parameters alone (LuxI or cell density). One can safely assume that at constant LuxI, the amount of AI produced is linearly proportional to cell density and vice-versa. Hence, <br />
<br />
<nowiki> AI = a LuxI * ?</nowiki><br />
<nowiki><br />
<br />
where, a --> constant<br />
? --> cell density</nowiki><br />
<br />
To prove the scaling argument, we find the CFP intensities corresponding to a particular concentration of AI at various aTc concentrations and cell densities. Subsequently, we verify that when the product of cell density and concentration of LuxI is the same in 2 trials, the amount of AI being produced is also the same.<br />
<br />
<br />
=='''Characterisation of Network 2'''==<br />
<br />
The plot for the open loop system in this case can be obtained from the open-loop system of Network 1 by fixing IPTG as the parameter and aTc as the variable input, so that we get the following family of curves (Fig. 10).<br />
[[Image:LUXICFPCFP_CURVES.jpg|thumb|200px|center|Fig.10 Plot of CFP against LuxI.CFP]]<br />
<br />
The steady states can then be obtained as described previously.<br />
<br />
<br />
== '''The Mathematical Model''' ==<br />
The Mathematical model for our system is available [[Media:Model.pdf|here]].<br />
<br />
The following links contain 2 derivations whose results have been used in the model.<br />
*[[Media:derivation_ai.pdf|[AI] Derivation]]<br />
*[[Media:derivation_luxrstar.pdf|[LuxR*] Derivation]]<br />
<br />
=='''References'''==<br />
<br />
1. Christopher M. and Bonnie L. (2005). Quorum Sensing: Cell-to-Cell Communication in Bacteria. '''Annu. Rev. Cell Dev. Biol.''', 21, 319–46.<br />
<br />
2. David Angeli, James E. Ferrell, Jr., and Eduardo D. Sontag (2004). Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems. '''PNAS''', 101, 1822 – 1827.</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-10T04:58:23Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|width="37%"<br />
|-<br />
|align="center"|'''The NCBS iGEM 2007 Experiments'''<br />
|-<br />
|align="justify"|<br />
<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
*pT.luxI.Cfp<br />
*pL.luxI.Cfp<br />
*pL.luxR.Yfp<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-08-10T04:55:04Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|width="37%"<br />
|-<br />
|align="center"|'''The NCBS iGEM 2007 Experiments'''<br />
|-<br />
|align="justify"|<br />
<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
Note that in flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. Click [[Media:Analysis.pdf|here]] for details about this mathematical tool for correction.</font><br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
<br />
<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
*pT.luxI.Cfp<br />
*pL.luxI.Cfp<br />
*pL.luxR.Yfp<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-07-15T07:36:36Z<p>Skundu: /* Experiment */</p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|width="35%"<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|-<br />
|align="justify"|<br />
<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
In flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. The details about this mathematical tool for correction can be found [[Media:Analysis.pdf|here]].</font><br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
*pL.Cfp<br />
*pT.luxI.Cfp<br />
*pL.luxI.Cfp<br />
*pL.luxR.Yfp<br />
<br />
=== Open loops ===<br />
<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)<br />
*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)<br />
<br />
=== Closed loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-07-14T06:45:53Z<p>Skundu: /* Experiment */</p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|width="35%"<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|-<br />
|align="justify"|<br />
<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
In flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. The details about this mathematical tool for correction can be found [[Media:Analysis.pdf|here]].</font><br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===<br />
<br />
=== Open loops ===</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-07-14T06:45:20Z<p>Skundu: /* Experiment */</p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|width="35%"<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|-<br />
|align="justify"|<br />
<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
In flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. The details about this mathematical tool for correction can be found [[Media:Analysis.pdf|here]].</font><br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
<br />
== Experiment ==<br />
<br />
=== Equivalences ===</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-07-14T06:43:30Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|width="35%"<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|-<br />
|align="justify"|<br />
<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
In flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. The details about this mathematical tool for correction can be found [[Media:Analysis.pdf|here]].</font><br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
<br />
<br />
<br />
==Experiment==</div>Skunduhttp://2007.igem.org/wiki/index.php/ExperimentsExperiments2007-07-14T06:39:31Z<p>Skundu: </p>
<hr />
<div>[[Image:Ncbs_Logo.jpg|100px|right]]<br />
[[Image:Ncbs.jpg|right]]<br />
{|<br />
|-<br />
|align="center"|The official wiki of the NCBS iGEM 2007 Team<br />
|}<br />
{| width="62%" align="right"<br />
! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]<br />
|}<br />
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"<br />
![[Bangalore|Bangalore]]<br />
![[The Company|The Team]]<br />
![[The Mission|The Mission]]<br />
![[Experiments|Experiments]]<br />
![[e-Notebook|e-Notebook]]<br />
|}<br />
<br />
{|<br />
|-<br />
|align="justify"|<br />
|<font size="2" face="Bookman Old Style"><br />
The following is the record of all the experiments done by us, each followed by graphs obtained by analysis of the corresponding microscopy and flow cytometry data.<br />
<br />
In flow cytometry a signal obtained from a filter does not exactly correspond to CFP or YFP amount inside a cell; when cells express both the proteins. This is because of spectral overlap of their excitation and emission spectra. We came up with a mathematical method to separate CFP and YFP from autofluorescence and noise. The details about this mathematical tool for correction can be found [[Media:Analysis.pdf|here]].</font><br />
|}<br />
<br />
<br />
<br />
<br />
<br />
==Experiment==</div>Skunduhttp://2007.igem.org/wiki/index.php/July_9July 92007-07-11T19:38:26Z<p>Skundu: /* Experiments */</p>
<hr />
<div>Back to ''[[Bangalore]]''<br />
<br />
Back to ''[[e-Notebook]]''<br />
<br />
==Experiments==<br />
<br />
== Analysis ==</div>Skundu