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Team:Paris Saclay/Notebook/August/30
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Revision as of 18:05, 28 September 2013
Notebook : August 30
Lab work
A - Aerobic/Anaerobic regulation system
Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006
1 - Results of liquid culture of Pfnr-RBS-Amil CP-Term in PSB1C3 in aerobic and anaerobic conditions
XiaoJing
|
IT WORKS !!! |
In anaerobic conditions, thank to promotor Pfnr, RBS-Amil CP-Term gene is activated and we can see a violet coloration of bacterias.
In aerobic conditions, promotor Pfnr is inactivated, RBS-Amil CP-Term isn't activated and we can't see any coloration of bacterias.
2 - Results of culture of Pfnr-RBS-Amil CP-Term in PSB1C3, NirB with RBS-LacZ-Term in PSB1C3 in aerobic and anaerobic conditions
XiaoJing
|
Purification of 08/22/13 didn't work. We have blue colonies for Pfnr with RBS-Amil CP-Term in PSB1C3 in aerobic and anaerobic conditions. We also have blue colonies for NirB with RBS-LacZ-Term in PSB1C3 in anaerobic conditions. |
3 - Ligation of Pfnr with RBS-LacZ-Term in PSB1C3
XiaoJing
Used quantities :
- Pfnr : 8µL
- RBS-LacZ-Term in PSB1C3 : 3µL
- Buffer ligation : 2µL
- Ligase : 1µL
- H2O : 6µL
4 - Transformation of ligation of Pfnr with RBS-LacZ-Term in PSB1C3 in DH5α
XiaoJing
Protocol : Bacterial transformation
We incubate it at 37°C with Xgal in aerobic conditions.
Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3
1 - Ligation of Pfnr with RBS-LacZ-Term in PSB3K3 and NarK with RBS-LacZ-Term in PSB3K3
XiaoJing
Used quantities :
- Pfnr, NarK : 3µL
- RBS-LacZ-Term : 3µL
- PSB3K3 : 5µL
- Buffer ligase : 2µL
- Ligase : 1µL
- H2O : 6µL
2 - Transformation of ligation of Pfnr with RBS-LacZ-Term in PSB3K3 and NarK with RBS-LacZ-Term in PSB3K3 in DH5α
XiaoJing
Protocol : Bacterial transformation
We incubate it at 37°C with Xgal in anaerobic conditions for NarK with RBS-LacZ-Term in PSB3K3. We incubate it at 37°C with Xgal in aerobic conditions for NarK with Pfnr-LacZ-Term in PSB3K3.
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FNR and BphR2 proteins
1 - Electrophoresis of Colony PCR of FNR, RBS-FNR and RBS-BphR2
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Expect sizes :
- FNR : 1096 bp
- RBS-FNR : 1014 bp
- RBS-BphR2 : 1469 bp
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We obtain fragment at right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good. |
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