Team:Paris Saclay/Notebook/August/29

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Notebook : August 29

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000 and BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Purification of colonies: NirB with RBS-LacZ-Term in pSB1C3, Pfnr with RBS-AmilCP-Term in pSB1C3 by streaking colonies in aerobic or anaerobic conditions

XiaoJing

Purification of 08/28/13 didn't work. We have blue colonies for Pndh* with RBS-AmilCP-Term in pSB1C3 in aerobic and anaerobic conditions. We also have blue colonies for NirB with RBS-LacZ-Term in pSB1C3 in anaerobic conditions. We will streak these colonies again.

PsPfnr2908.jpg PsNirB2908.jpg

We streak colonies from construction :

  • Pndh* with RBS-Amil CP-Term in pSB1C3 with O2 at 37°C
  • Pndh* with RBS-Amil CP-Term in pSB1C3 without O2 at 37°C
  • Pndh* with RBS-Amil CP-Term in pSB1C3 with O2 at 30°C
  • Pndh* with RBS-Amil CP-Term in pSB1C3 without O2 at 30°C
  • NirB with RBS-LacZ-Term in pSB1C3 with O2 with Xgal at 37°C
  • NirB with RBS-LacZ-Term in pSB1C3 without O2 with Xgal at 37°C

We also purify Pndh* with RBS-AmilCP-Term in pSB1C3 in liquid culture at 37°C using :

  • Pndh* with RBS-AmilCP-Term in pSB1C3 in aerobic conditions :
    • LB : 10 mL
    • Clone : 1 and 2
  • Pndh* with RBS-AmilCP-Term in pSB1C3 in anaerobic conditions :
    • LB : 50mL
    • Clone : 1 and 2

2 - PCR Colony of strain DH5α containing plasmid pSB1C3 with Pndh* and RBS_AmilCP-Term

XiaoJing

We resuspend our colonies in 20µL of H2O.

Used quantities :

  • DNA : 2µL of resuspend colony
  • PCR mix: 23µL

PCR mix:

    • Oligo 43 : 14µL
    • Oligo 44 : 14µL
    • dNTP : 14µL
    • Buffer Dream Taq : 69µL
    • Dream Taq : 5.5µL
    • H2O : 577µL

PsPCR2908.jpg

3 - Digestion of BBa_K1155000, BBa_K1155003, BBa_K1155007 by Xbal/PstI

XiaoJing

We used clone 9 and 12 for BBa_K1155003, clone 10, 11 and 15 for BBa_K1155007

Used quantities :

  • BBa_K1155003, BBa_K1155007
    • DNA : 14µL
    • Buffer FD : 2µL
    • XbaI FD : 2µL
    • PstI FD : 2µL
  • BBa_K1155000 :
    • DNA : 5µL
    • Buffer FD : 2µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • H2O : 9µL

We incubatethe digestion for 30 minutes at 37°C.

4 - Electrophoresis to check the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI

XiaoJing

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of RBS-AmilCP-Term clone 9 + 1µL of 6X loading dye
  • Well 3 : 5µL of RBS-AmilCP-Term clone 12 + 1µL of 6X loading dye
  • Well 4 : 5µL of RBS-LacZ-Term clone 10 + 1µL of 6X loading dye
  • Well 5 : 5µL of RBS-LacZ-Term clone 11 + 1µL of 6X loading dye
  • Well 6 : 5µL of RBS-LacZ-Term clone 15 + 1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • RBS-LacZ-Term : 3500bp
  • RBS-AmilCP-Term : 824bp
[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 3 : 5µL of Pndh* + 1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • Pndh* : 111bp

We obtain fragments at the right size. We will purify them.

5 - Gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI

XiaoJing

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • Pndh* : 26.2ng/µL

6 - Electrophoresis to check the gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI

Damir

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 2µL of RBS-LacZ-Term clone 10 + 1µL of 6X loading dye
  • Well 3 : 2µL of RBS-AmilCP-Term clone 9 + 1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • RBS-LacZ-Term : 3500bp
  • RBS-Amil CP-Term : 824bp

We obtain fragments at the right size for RBS-LacZ-Term. We will ligate it.

7 - Electrophoresis to check the digestion of BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI

XiaoJing

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of PNirB clone 7 + 1µL of 6X loading dye
  • Well 3 : 5µL of NarG clone 6 + 1µL of 6X loading dye
  • Well 4 : 5µL of BBa_K1155006 already digested by SpeI + 1µL of 6X loading dye
  • Well 5 : 5µL of BBa_K1155004 already digested by SpeI + 1µL of 6X loading dye
  • Well 5 : 5µL of BBa_K1155005 already digested by SpeI + 1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • NarK, NarG, NirB : 200bp

We obtain fragments at the right size for NarK. We will ligate it

Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3

1 - Digestion of BBa_J04450 by EcoRI/PstI

Anaïs

Used quantities :

  • Buffer FD: 2µL
  • H2O : 5µL
  • DNA : 9µL
  • EcoRI FD : 1µL
  • PstI FD : 1µL

We incubate the digestion at 37°C for 10 minutes.

2 - Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI

XiaoJing

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 3 : 5µL of PSB3K3 + 1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • pSB3K3 : 2750bp

We obtain fragments at the right size. We will ligate it.

3 - Gel purification of the digestion of BBa_J04450 by PstI/SpeI

XiaoJing

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • Pndh* : 7.2ng/µL

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FRN and BphR2 proteins

1 - PCR Colony of FNR, RBS-FNR and RBS-BphR2 in DH5α strain

XiaoJing

Transformation of 08/28/13 works. We will do a Colony PCR.

We mix our colonies in 20µL of H2O.

Used quantities :

  • DNA : 2µL
  • Mix : (it was divided in 8 tubes for 8 different colonies for each assembly with 23µL of mix in each tube. We do it twice.)
    • Oligo 43 : 27.5µL
    • Oligo 44 : 27.5µL
    • dNTP : 27.5µL
    • Buffer Dream Taq : 137.5µL
    • Dream Taq : 11µL
    • H2O : 1144µL

PsPCR2908.jpg

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