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

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 with RBS-Amil CP-Term in PSB1C3, NirB with RBS-LacZ-Term in PSB1C3 in aerobic and anaerobic conditions

XiaoJing

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.

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

Expect sizes :

• FNR : 1096 bp
• RBS-FNR : 1014 bp
• RBS-BphR2 : 1469 bp