Experiments

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|align="center"|<font size="2" face="Garamond">The official wiki of the NCBS iGEM 2007 Team</font>
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|rowspan="3" width="62%" |[[Image:Ncbs_Logo.jpg|100px|right]]
[[Image:Ncbs.jpg|right]]
[[Image:Ncbs.jpg|right]]
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{|
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|-
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|align="center"|'''The NCBS iGEM 2007 Experiments'''
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|align="center"|The official wiki of the NCBS iGEM 2007 Team
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| align="justify"|<font size="2" face="Bookman Old Style">
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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.
 +
 
 +
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>
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{| width="62%" align="right"
{| width="62%" align="right"
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! [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]
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! align="center"|[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]
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{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"
{| style="color:#294e9c;background-color:#e1e0db;" cellpadding="3" cellspacing="1" border="1" bordercolor="#1100ff" width="62%" align="right"
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![[Bangalore|Bangalore]]
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!align="center"|[[Bangalore|Bangalore]]
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![[The Company|The Team]]
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!align="center"|[[The Company|The Team]]
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![[The Mission|The Mission]]
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!align="center"|[[The Mission|The Mission]]
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![[Experiments|Experiments]]
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!align="center"|[[Experiments|Experiments]]
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![[e-Notebook|e-Notebook]]
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!align="center"|[[e-Notebook|e-Notebook]]
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{| align="center"
 
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|-
 
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|'''The Bangalore iGEM Journal, 07'''
 
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== Experiment ==
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=== Equivalences ===
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*pL.Cfp
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{|
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|- align="justify"
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|[[Image:pLC_dup.png|400px]]
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|Details...
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|}
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*pT.luxI.Cfp
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{|
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|- align="justify"
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|[[Image:pTIC_dup.png|400px]]
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|Details...
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|}
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*pL.luxI.Cfp
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{|
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|- align="justify"
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|[[Image:pLIC_dup.png|400px]]
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|Details...
|}
|}
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=='''Protocols''' ==
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*pL.luxR.Yfp
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{|
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|- align="justify"
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|[[Image:pLRY_dup.png|400px]]
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|Details...
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|}
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=== Open loops ===
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'''1) Induction experiments:'''
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*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)
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  Grow in LB for 10 hours
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{|
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  Then, induce in Glu M9 for 12 hours at various concns of IPTG/aTc
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|- align="justify"
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                +ve control: pL YFP in 50,100 uM IPTG
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|[[Image:Openloop_0.png|400px]]
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                -ve control: K12z1   
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|Details...
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  Take microscopy images
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|}
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'''2) Open loop experiments:'''
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  i) Morning imaging (followed usually)
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      Day 0: 0000: Inoculate pTLuxI.C in LB
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      Day 1: 0100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]
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            1230: Inoculate pLLuxR.Y pRC in LB
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            2200: Filter required OD flask
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            2230: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9
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                          -ve control: K12z1   
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      Day 2: 1000: Take microscopy images
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  ii) Night imaging
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      Day 1: 1100: Inoculate pTLuxI.C in LB
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            2100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]
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            2330: Inoculate pLLuxR.YpRC in LB
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      Day 2: 0900: Filter required OD flask
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            0930: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9
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                          -ve control: K12z1 in 50 old:50 new 2xM9
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            2130: Take microscopy images
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==Notes==
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*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)
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*All inductions with IPTG: 0,5,10,50,100,500,1000 uM
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{|
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|- align="justify"
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*All inductions with  aTc: 0,1,5,10,20,50 ng/mL
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|[[Image:Openloop_1.png|400px]]
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|Details...
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|}
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*The open loop experiments to be carried out with following aTc concentrations:
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*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)
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{|
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|- align="justify"
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|[[Image:Openloop_5.png|400px]]
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|Details...
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|}
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[A] : 20 ng/ml
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*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)
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{|
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[B] : 10 ng/ml
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|- align="justify"
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|[[Image:Openloop_10.png|400px]]
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[C] : 5 ng/ml
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|Details...
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[D] : 1 ng/ml
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[E] : 50 ng/ml
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[F] : 0 ng/ml
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[AA] : 20 ng/ml (repeat A)
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[FF] : 0 ng/ml (repeat F)
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The '''''DUPLICATES''''' for each of the above experiments are denoted by [Aa], [Bb], [Cc], [Dd], [Ee] and [Ff] respectively.
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== Experiments Record ==
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*Calibration of the number of colonies obtained at different optical densities.
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*Transformation of competent E.coli (strain K12Z1) cells with constructs.
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*'''Induction of the construct pLac cfp''' 
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E.coli cells transformed with the construct pLac cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.
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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.
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{| cellpadding="2" cellspacing="3" border="1"
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!Sample
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!100mM IPTG (ul)
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!1mM IPTG (ul)
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!Inoculum volume from LB (ul)
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!Volume of  M9 added (ml)
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|-align="center"
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|pLac cfp (0 uM)
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| -
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| -
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|3 ul
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|3 ml
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|-align="center"
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|pLac cfp (1 uM)
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| -
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|3 ul
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|3 ul
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|3 ml
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|-align="center"
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|pLac cfp (10 uM)
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| -
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|30 ul
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|3 ul
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|3 ml
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|-align="center"
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|pLac cfp (50 uM)
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|1.5 ul
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| -
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|3 ul
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|3 ml
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|-align="center"
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|pLac cfp (100 uM)
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|3 ul
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| -
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|3 ul
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|3 ml
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|-align="center"
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|pLac cfp (1000 uM)
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|30 ul
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| -
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|3 ul
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|3 ml
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|-align="center"
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|K12Z1 (Autoflourescence)
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| -
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| -
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|3 ul
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|3 ml
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|}
|}
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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.
 
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'''Results:'''
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*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)
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The mean fluorescence of CFP was seen to increase with increasing concentrations of the IPTG.
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{|
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|- align="justify"
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*'''Induction of the construct pLac yfp'''
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|[[Image:Openloop_20.png|400px]]
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E.coli cells transformed with the construct pLac yfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.
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|Details...
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+
-
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.
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{| cellpadding="2" cellspacing="3" border="1"
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!Sample
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!100mM IPTG (ul)
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!1mM IPTG (ul)
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!Inoculum volume from LB (ul)
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!Volume of  M9 added (ml)
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|-align="center"
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|pLac yfp (0 uM)
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| -
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| -
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|3 ul
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|3 ml
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|-align="center"
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|pLac yfp (1 uM)
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| -
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|3 ul
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|3 ul
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|3 ml
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|-align="center"
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|pLac yfp (10 uM)
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| -
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|30 ul
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|3 ul
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|3 ml
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|-align="center"
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|pLac yfp (50 uM)
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|1.5 ul
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| -
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|3 ul
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|3 ml
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|-align="center"
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|pLac yfp (100 uM)
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|3 ul
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| -
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|3 ul
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|3 ml
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|-align="center"
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|pLac yfp (1000 uM)
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|30 ul
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| -
+
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|3 ul
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|3 ml
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|-align="center"
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|K12Z1 (Autoflourescence)
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| -
+
-
| -
+
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|3 ul
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|3 ml
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|}
|}
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'''Results:'''
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*pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)
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The mean fluorescence of YFP was seen to increase with increasing concentrations of the IPTG.
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{|
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|- align="justify"
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*'''Induction of the construct pTet.luxI.cfp''' 
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|[[Image:Openloop_50.png|400px]]
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E.coli cells transformed with the construct pTet.luxI.cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.
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|Details...
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+
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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.
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{| cellpadding="2" cellspacing="3" border="1"
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!Sample
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!10ug/ml aTc (ul)
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!Inoculum volume from LB (ul)
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!Volume of  M9 added (ml)
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|-align="center"
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|pTet luxI cfp(0 ng/ml)
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| -
+
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|3 ul
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|3 ml
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|-align="center"
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|pTet luxI cfp(1 ng/ml)
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|0.3 ul
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|3 ul
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|3 ml
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|-align="center"
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|pTet luxI cfp(10 ng/ml)
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|3 ul
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|3 ul
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|3 ml
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|-align="center"
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|pTet luxI cfp(25 ng/ml)
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|7.5 ul
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|3 ul
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|3 ml
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|-align="center"
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|pTet luxI cfp(50 ng/ml)
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|15 ul
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|3 ul
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|3 ml
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|-align="center"
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|pTet luxI cfp(100 ng/ml)
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|30 ul
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|3 ul
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|3 ml
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|-align="center"
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|K12Z1 (Autoflourescence)
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-
| -
+
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|3 ul
+
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|3 ml
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|}
|}
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'''Results:'''
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=== Closed loops ===
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The mean fluorescence of CFP was seen to increase with increasing concentrations of aTc.
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*'''Open Loop Trial'''
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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.
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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.
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*'''The Scaling Argument'''
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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.
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== Discussions ==
+

Latest revision as of 15:51, 24 August 2007

The official wiki of the NCBS iGEM 2007 Team
Ncbs Logo.jpg
Ncbs.jpg
The NCBS iGEM 2007 Experiments

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.

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 here for details about this mathematical tool for correction.

National Centre for Biological Sciences, Bangalore


Bangalore The Team The Mission Experiments e-Notebook


Contents

Experiment

Equivalences

  • pL.Cfp
PLC dup.png Details...
  • pT.luxI.Cfp
PTIC dup.png Details...
  • pL.luxI.Cfp
PLIC dup.png Details...
  • pL.luxR.Yfp
PLRY dup.png Details...

Open loops

  • pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (0 ng/ml aTc)
Openloop 0.png Details...
  • pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (1 ng/ml aTc)
Openloop 1.png Details...
  • pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (5 ng/ml aTc)
Openloop 5.png Details...
  • pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (10 ng/ml aTc)
Openloop 10.png Details...
  • pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (20 ng/ml aTc)
Openloop 20.png Details...
  • pT.luxI.Cfp::pL.luxR.Yfp.pR.Cfp (50 ng/ml aTc)
Openloop 50.png Details...

Closed loops

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