Team:Paris Saclay/Notebook/August/21

From 2013.igem.org

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
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===='''Obtaining FNR Promoter (nirB,narG,narK)plus  RBS_LacZ+Term_PSB1C3 or  RBS_Amicip+Term_PSB1C3 '''====
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===='''Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
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=====1 - Colony PCR on e.coli with FNR Promoter (nirB,narG,narK)plus  RBS_LacZ+Term_PSB1C3 or RBS_AmilCP+Term_PSB1C3  for 8 colonies of each=====
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=====1 - Colony PCR of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α=====
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Damir,Xiaojing
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*A1-8: FNR Promoter nirB plus  RBS_LacZ+Term_PSB1C3
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Damir, Xiaojing
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*B1-8: FNR Promoter nirB plus  RBS_AmilCP+Term_PSB1C3
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*C1-8: FNR Promoter narG plus  RBS_LacZ+Term_PSB1C3
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*D1-8: FNR Promoter narG plus  RBS_AmilCP+Term_PSB1C3
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*E1-8: FNR Promoter narK plus  RBS_LacZ+Term_PSB1C3
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*F1-8: FNR Promoter narK Plus  RBS_AmilCP+Term_PSB1C3
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*Picking of 48 colonies
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*Preparation  Master mix A
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Colonies repiquée dans 10µL d'eau.
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**H<sub>2</sub>O : 1880µL
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We do 8 PCR for each ligation.
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**dNTP : 50µL
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**VR primer : 50µL
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**DreamTaq buffer 10x : 250µL
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**DreamTaq enzyme : 20µL
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*Preparation  Master mix B
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**Master mix A :1125µL
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**VF2 primer : 25µL
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 +
Used quantities :
 +
* Mix A :
 +
** Buffer Dream Taq : 250µL
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** dNTP : 50µL
 +
** Oligo 44 : 50µL
 +
** Dream Taq : 20µL
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** H2O : 1.88mL
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Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]] By using  Master mix B
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* NirB with RBS-LacZ-Term in PSB1C3 :  
 +
** 45µL of Mix A+1µL of oligo 45+2µL of DNA
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* 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
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** Oligo 43 : 25µL
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* 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
=====2 - Gel electrophoresis of the colony PCR products=====
=====2 - Gel electrophoresis of the colony PCR products=====

Revision as of 15:21, 17 September 2013

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Contents

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
2 - Gel electrophoresis of the colony PCR products
[[]]
  • 6µL DNA Ladder
  • 10µL sample per well
  • Gel : 0.8%

Expected size : 3583bp

3 - Culture

Nguyen

  • BBa_K115500 strains (FNR repressor)in 5ml LB with Chlorenphenicol
  • strain MG1665 Δ fnr:km clone1 in 5ml LB with kanamicine

Incubate 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
  • GFP : ... NON VISIBLE SUR LE GEL ???????

We obtain a fragment at the right size. We can purify it.


A - Aerobic/Anaerobic regulation system / B - PCB se nsing system

1 - Gibson assembloy.

Xiaojing

  • RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
  • FNR_part1, FNR part1 and plasmid PSB1C3
  • RBS_FNR part1, FNR_part2 and plasmid PSB1C3


Sample Volume:

  • Tube 1 : BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
  • Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
  • Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)

These 3 mixture is incubated at 50°C for up to one hour. Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.




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