Contents 1 Notebook : August 29 1.1 Lab work 1.1.1 A - Aerobic/Anaerobic regulation system 1.1.1.1 Objective : characterize BBa_K1155000 and BBa_K1155004, BBa_K1155005, BBa_K1155006 1.1.1.2 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 1.1.1.3 2 - PCR Colony of strain DH5α containing plasmid pSB1C3 with Pndh* and RBS_AmilCP-Term 1.1.1.4 3 - Digestion of BBa_K1155000, BBa_K1155003, BBa_K1155007 by Xbal/PstI 1.1.1.5 4 - Electrophoresis to check the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI 1.1.1.6 5 - Gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI 1.1.1.7 6 - Electrophoresis to check the gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI 1.1.1.8 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 1.1.1.9 8 - Purification colony of strain MG1655Z1 Δfnr 1.1.1.10 Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3 1.1.1.11 1 - Digestion of BBa_J04450 by EcoRI/PstI 1.1.1.12 2 - Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI 1.1.1.13 3 - Gel purification of the digestion of BBa_J04450 by PstI/SpeI 1.1.2 A - Aerobic/Anaerobic regulation system / B - PCB sensor system 1.1.2.1 Objective : obtaining FRN and BphR2 proteins 1.1.2.2 1 - PCR Colony of FNR, RBS-FNR and RBS-BphR2 in DH5α strain

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.

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

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

Expected sizes :

• Pndh* : 111bp

We obtained 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 obtained 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 6 : 5µL of BBa_K1155005 already digested by SpeI + 1µL of 6X loading dye Gel : 1.0%

Expected sizes :

• NarK, NarG, NirB : 2000bp

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

8 - Purification colony of strain MG1655Z1 Δfnr

XiaoJing

Culture from 08/28/13 works. We will do a purification of one colonies on plates with kanamycin, ampicilin and chloramphenicol antibiotics.

We streaked 4 colonies in each plate but we used the same colony to streak on plates with LB or kanamycin or ampicilin or chloramphenicol antibiotics. We incubate our colonies at 42°C.

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 obtained 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

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