Team:Paris Saclay/Notebook/August/30

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IT WORKS !!!
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Revision as of 15:59, 4 October 2013

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Contents

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 !!!

PsPfnr3008.jpg

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

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.

PsPfnrcult3008.jpg PsNirBcult3008.jpg

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

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 20µL of Pfnr with RBS_AmilCP-Term clone 1+4µL of 6X loading dye
  • Well 3 : 20µL of Pfnr with RBS_AmilCP-Term clone 2+4µL of 6X loading dye
  • Well 4 : 20µL of Pfnr with RBS_AmilCP-Term clone 3+4µL of 6X loading dye
  • Well 5 : 20µL of Pfnr with RBS_AmilCP-Term clone 4+4µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • Pfnr with RBS_AmilCP-Term : 1000bp

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

]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 to 17 : 5µL of FNR+1µl of 6X loading dye
  • Well 18 to 26 : 5µL of RBS-BphR2+1µl of 6X loading dye
  • Well 27 : 6µL DNA Ladder
  • Well 28 to 41 : 5µL of RBS-FNR+1µl of 6X loading dye
  • Well 42 to 49 : 5µL of RBS-BphR2+1µl of 6X loading dye
  • WEll 50 : 6µL DNA Ladder
  • Gel : 1%

Expect sizes :

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

We obtain fragment at right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good.


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