E-Notebook
From 2007.igem.org
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| [http://www.ncbs.res.in/ National Centre for Biological Sciences, Bangalore] |
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| Bangalore | The Company | The Mission | e-Notebook |
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| The Bangalore iGEM Journal, 07
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Contents |
e-notebook
| May 07 | June 07 | July 07 | ||||||||||||||||||||||||||||||||
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| S | M | T | W | T | F | S | S | M | T | W | T | F | S | S | M | T | W | T | F | S | ||||||||||||||
| 1 | 2 | 3 | 4 | 5 | 1 | 2 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | |||||||||||||||||||||
| 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.
This Week
Tue, June 26: pL LuxI.C; full range IPTG induction (for equivalence)
Wed, June 27: pL LuxR.Y; full range IPTG induction (for equivalence) [morn]
Wed, June 27: pT LuxI.C; full range aTc induction (for equivalence) [eve]
Thu, June 28: pL C; full range IPTG induction (for equivalence) [morn]
Thu, June 28: Open loop experiment with aTC=20 ng/mL, IPTG=full range [eve]
Fri, June 29: Open loop experiment with aTC=50,10 ng/mL, IPTG=full range [morn/eve]
Sat, June 30: Open loop experiment with aTC=5 ng/mL, IPTG=full range [morn]
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
