Notebook : August 21

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Colony PCR of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α

Damir, Xiaojing

Colonies repiquée dans 10µL d'eau. We do 8 PCR for each ligation.

Used quantities :

• Mix A :
  • Buffer Dream Taq : 250µL
  • dNTP : 50µL
  • Oligo 44 : 50µL
  • Dream Taq : 20µL
  • H2O : 1.88mL

• • • NirB with RBS-LacZ-Term in pSB1C3 :
      • 45µL of Mix A+1µL of oligo 45+2µL of DNA
    • NirB with RBS-Amil CP-Term in pSB1C3 :
      • 45µL of Mix A+1µL of oligo 45 +2µL of DNA

• • • NarG with RBS-LacZ-Term in pSB1C3 :
      • 45µL of Mix A+1µL of oligo 41+2µL of DNA
    • NarG with RBS-Amil CP-Term in pSB1C3 :
      • 45µL of Mix A+1µL of oligo 41 +2µL of DNA

• • • NarK with RBS-LacZ-Term in pSB1C3 :
      • 45µL of Mix A+1µL of oligo 47+2µL of DNA
    • NarK with RBS-Amil CP-Term in pSB1C3 :
      • 45µL of Mix A+1µL of oligo 47 +2µL of DNA

• Mix B :
  • Mix A : 1.125mL
  • Oligo 43 : 25µL

• • • NirB with RBS-LacZ-Term in pSB1C3 :
      • 23µL of Mix B+2µL of DNA
    • NirB with RBS-Amil CP-Term in pSB1C3 :
      • 23µL of Mix B+2µL of DNA

• • • NarG with RBS-LacZ-Term in pSB1C3 :
      • 23µL of Mix B+2µL of DNA
    • NarG with RBS-Amil CP-Term in pSB1C3 :
      • 23µL of Mix B+2µL of DNA

• • • NarK with RBS-LacZ-Term in pSB1C3 :
      • 23µL of Mix B+2µL of DNA
    • NarK with RBS-Amil CP-Term in pSB1C3 :
      • 23µL of Mix B+2µL of DNA

PCR Program for NirB with RBS-LacZ-Term in pSB1C3, NarG with RBS-LacZ-Term in pSB1C3, NarK with RBS-LacZ-Term in pSB1C3 :

PCR Program for NirB with RBS-Amil CP-Term in pSB1C3, NarG with RBS-Amil CP-Term in pSB1C3, NarK with RBS-Amil CP-Term in pSB1C3 :

2 - Culture of BBa_K1155000

Nguyen

We made culture of BBa_K115500 strains in 5ml LB with Chlorenphenicol. We incubate it at 37°C over night with shake 180 rpm.

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

1 - Electrophoresis of the digestion of BBa_J04450 by EcoRI/PstI

XiaoJing

Well 1 : 6µL DNA Ladder Well 2 : 17µL of BBa_J04450 digested by EcoRI/PstI+3µL of H2O+4µL of 6X loading buffer Gel : 1%

Expected sizes :

• PSB3K3 : 2750 bp

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

Objective : obtaining FNR and BphR2 proteins

1 - Transformation of RBS-BphR2, FNR, RBS-FNR in DH5α

XiaoJing

Protocol : Bacterial transformation