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From 2007.igem.org

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Protocols

1) Induction experiments:

  Grow in LB for 10 hours
  Then, induce in Glu M9 for 12 hours at various concns of IPTG/aTc
               +ve control: pL YFP in 50,100 uM IPTG 
               -ve control: K12z1     
  Take microscopy images

2) Open loop experiments:

  i) Morning imaging
     Day 0: 0000: Inoculate pTLuxI.C in LB
     Day 1: 1000: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]
            1230: Inoculate pLLuxR.Y pRC in LB
            2200: Filter required OD flask
            2230: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9
                          -ve control: K12z1    
     Day 2: 1030: Take microscopy images
  ii) Night imaging
     Day 1: 1100: Inoculate pTLuxI.C in LB
            2100: Transfer to Glu M9 in 250 mL flask with a particular [aTc] and inoculum [x3]
            2330: Inoculate pLLuxR.YpRC in LB
     Day 2: 0900: Filter required OD flask
            0930: Make 7 dilutions for pLLuxR.YpRC with 50 old:50 new 2xM9
                          -ve control: K12z1 in 50 old:50 new 2xM9
            2130: Take microscopy images

Experiments done

• Calibration of the number of colonies obtained at different optical densities.
• Transformation of competent E.coli (strain K12Z1) cells with constructs.
• Induction of the construct pLac cfp

E.coli cells transformed with the construct pLac cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.

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.

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.

Results: The mean fluorescence of CFP was seen to increase with increasing concentrations of the IPTG.

• Induction of the construct pLac yfp

E.coli cells transformed with the construct pLac yfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.

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.

Results: The mean fluorescence of YFP was seen to increase with increasing concentrations of the IPTG.

• Induction of the construct pTet luxI cfp

E.coli cells transformed with the construct pTet luxI cfp were cultured for 10 hours in Luria Bertani medium containing 50mg/ml Ampicillin.

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.

Results: The mean fluorescence of CFP was seen to increase with increasing concentrations of aTc.

• Open Loop Trial

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.

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 frown 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.

Two sets of experiments were carried out , where cells were grown to an optical density of 0.04 and 0.4 respectively in order to check for density dependence of autoinducer production. The cells were then imaged using a phase contrast microscope.

Results: Density dependence of autoinducer production was observed as fluorescence levels in the sample induced with 100uM IPTG and grown to an optical density of 0.4 was greater than fluorescence levels in a corresponding sample grown to an optical density of 0.04.

This Week

 Mon, July 2: pL LuxI.C for IPTG-aTc equivalence (morn)
 Tue, July 3: pL LuxR.Y for cfp-yfp equivalence, 
              pT LuxI.C for IPTG-aTc
 Wed, July 4: Open loop [E],[F] (aTc) 
              At OD 0.1: To prove scaling argument for AI.
 Thu, July 5: aTC 10,20 ng/mL   [X,Y]
 DUPLICATES:
 ---------
 At OD 0.2: 
 Fri, July 6: Open loop: aTC 20,10 ng/mL  [A',B']  
 Sat, July 7: Open loop: aTC 5,1  ng/mL    [C',D']

Notes

• All inductions with IPTG: 0,5,10,50,100,500,1000 uM

• All inductions with aTc: 0,1,5,10,20,50 ng/mL

• The open loop experiments to be carried out with following aTc concentrations:

[A] : 20 ng/ml

[B] : 10 ng/ml

[C] : 5 ng/ml

[D] : 1 ng/ml

[E] : 50 ng/ml

[F] : 0 ng/ml

[AA] : 20 ng/ml (repeat A)

[FF] : 0 ng/ml (repeat F)

To-do List

DUPLICATE:

For equivalences:

Wed, July 4: pL C, pL LuxI.C (morn/eve)

Thu, July 5: pL LuxR.Y, pT LuxI.C (morn/eve)

Open loop:

At OD 0.2:

Fri, July 6: aTC 50,20 ng/mL (morn/eve)

Sat, July 7: aTC 10,5 ng/mL (morn/eve)

Sun, July 8: aTC 1,0 ng/mL (morn/eve)

At OD 0.1: (to prove scaling argument)

Mon, July 9: aTC 10,20 ng/mL (morn/eve)

<MAKE PREDICTIONS>

Closed loop:

Tue, July 10

Wed, July 11

Thurs/Fri, July 12/13: Wrap-up

Discussions