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Team:Paris Saclay/Notebook/August/30
From 2013.igem.org
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
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IT WORKS !!! |
In anaerobic conditions, the constitutive promotor Pndh is repressed, RBS_Amil CP-Term isn't expressed. We can't see any coloration of bacterias.
In aerobic conditions, the constitutive promotor Pndh isn't repressed, RBS-Amil CP-Term is expressed and we can see violet bacterias.
2 - Results of culture of Pfnr with RBS-Amil CP-Term in pSB1C3, NirB with RBS-LacZ-Term in pSB1C3 in aerobic and anaerobic conditions
XiaoJing
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Purification of 08/29/13 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.
5 - 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
6 - 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.
7 - Electrophoresis of Colony PCR of Pfnr with RBS_AmilCP-Term in DH5α
XiaoJing
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Expected sizes :
- Pfnr with RBS_AmilCP-Term : 1000bp
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We obtain fragments at the right size. |
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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