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1 Notebook : August 21
- 1.1 Lab work
  - 1.1.1 A - Aerobic/Anaerobic regulation system
    - 1.1.1.1 Obtaining FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus RBS_Amicip+Term_PSB1C3
      - 1.1.1.1.1 1 - Colony PCR on e.coli with FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus RBS_Amicip+Term_PSB1C3 for 8 colonies of each
      - 1.1.1.1.2 2 - Gel electrophoresis of the colony PCR products
  - 1.1.2 A - Aerobic/Anaerobic regulation system / B - PCB sensing system
    - 1.1.2.1 2 - Gibson assembly.
    - 1.1.2.2 3 - PCR pbaroduct (made the 08/01/2013) purification

Notebook : August 21

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus RBS_Amicip+Term_PSB1C3

1 - Colony PCR on e.coli with FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus RBS_Amicip+Term_PSB1C3 for 8 colonies of each

Damir,Xiaojing

A1-8: FNR Promoter nirB plus RBS_LacZ+Term_PSB1C3 B1-8: FNR Promoter nirB plus RBS_Amicip+Term_PSB1C3 C1-8: FNR Promoter narG plus RBS_LacZ+Term_PSB1C3 D1-8: FNR Promoter narG plus RBS_Amicip+Term_PSB1C3 E1-8: FNR Promoter narK plus RBS_LacZ+Term_PSB1C3 F1-8: FNR Promoter narK Plus RBS_Amicip+Term_PSB1C3

Picking of 48 colonies

Preparation Master mix A
- H₂O : 1880µL
- dNTP : 50µL
- VR primer : 50µL
- DreamTaq buffer 10x : 250µL
- DreamTaq enzyme : 20µL
Preparation Master mix B
- Master mix A :1125µL
- VF2 primer : 25µL

Protocol : Colony PCR By using Master mix B

PCR Program :

2 - Gel electrophoresis of the colony PCR products

Damir

[[]]	6µL DNA Ladder 10µL sample per well Gel : 0.8%

Expected size : 3583bp

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

2 - Gibson assembly.

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.

3 - PCR pbaroduct (made the 08/01/2013) purification

Damirr available quantity:

FNR Part1 : 10 µl
FNR Part2 : 19 µl
RBS FNR Part1 :16.1µl
RBS BphR2 Part1 : 28µl
BphR2 Part1 : 16.4 µl
BphR2 Part2 : 18.9 µl

Protocol : kit purification

Manipulation error : The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.

Team:Paris Saclay/Notebook/August/21