E-Notebook

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The official wiki of the NCBS iGEM 2007 Team

[http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore]

Bangalore	The Company	The Mission	e-Notebook

The Bangalore iGEM Journal, 07

e-notebook

May 07							June 07							July 07
S	M	T	W	T	F	S	S	M	T	W	T	F	S	S	M	T	W	T	F	S
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6	7	8	9	10	11	12	3	4	5	6	7	8	9	8	9	10	11	12	13	14
13	14	15	16	17	18	19	10	11	12	13	14	15	16	15	16	17	18	19	20	21
20	21	22	23	24	25	26	17	18	19	20	21	22	23	22	23	24	25	26	27	28
27	28	29	30	31			24	25	26	27	28	29	30	29	30

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.

Sample	100mM IPTG (ul)	1mM IPTG (ul)	Inoculum volume from LB (ul)	Volume of M9 added (ml)
pLac cfp (0 uM)	-	-	3 ul	3 ml
pLac cfp (1 uM)	-	3 ul	3 ul	3 ml
pLac cfp (10 uM)	-	30 ul	3 ul	3 ml
pLac cfp (50 uM)	1.5 ul	-	3 ul	3 ml
pLac cfp (100 uM)	3 ul	-	3 ul	3 ml
pLac cfp (1000 uM)	30 ul	-	3 ul	3 ml
K12Z1 (Autoflourescence)	-	-	3 ul	3 ml

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.

Sample	100mM IPTG (ul)	1mM IPTG (ul)	Inoculum volume from LB (ul)	Volume of M9 added (ml)
pLac yfp (0 uM)	-	-	3 ul	3 ml
pLac yfp (1 uM)	-	3 ul	3 ul	3 ml
pLac yfp (10 uM)	-	30 ul	3 ul	3 ml
pLac yfp (50 uM)	1.5 ul	-	3 ul	3 ml
pLac yfp (100 uM)	3 ul	-	3 ul	3 ml
pLac yfp (1000 uM)	30 ul	-	3 ul	3 ml
K12Z1 (Autoflourescence)	-	-	3 ul	3 ml

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.

Sample	10ug/ml aTc (ul)	Inoculum volume from LB (ul)	Volume of M9 added (ml)
pTet luxI cfp(0 ng/ml)	-	3 ul	3 ml
pTet luxI cfp(1 ng/ml)	0.3 ul	3 ul	3 ml
pTet luxI cfp(10 ng/ml)	3 ul	3 ul	3 ml
pTet luxI cfp(25 ng/ml)	7.5 ul	3 ul	3 ml
pTet luxI cfp(50 ng/ml)	15 ul	3 ul	3 ml
pTet luxI cfp(100 ng/ml)	30 ul	3 ul	3 ml
K12Z1 (Autoflourescence)	-	3 ul	3 ml

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.

The Week Ahead

The to-do List

For equivalences:

pL LuxI.C: Tue, June 26

pL LuxR.Y: Wed, June 27 (morn)

pT LuxI.C: Wed, June 27 (eve)

pL C: Thu, June 28 (morn)

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

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

Open loop:

At OD 0.2:

Thu, June 28: aTC 20 ng/mL (eve)

Fri, June 29: aTC 50,10 ng/mL (morn/eve)

Sat, June 30: aTC 5 ng/mL (morn)

Mon, July 2: aTC 1,0 ng/mL (morn)

At OD 0.1: (to prove scaling argument)

Tue, July 3: aTC 10,20 ng/mL (morn/eve)

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

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