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From 2013.igem.org

Contents 1 Notebook : August 30 1.1 summary 1.2 lab work 1.2.1 1 -Gel electrophoresis of PCR clonies on Gibson assembly . 1.2.2 2 -Gel electrophoresis of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 . 2 Notebook : August 23 2.1 Lab work 2.1.1 A - Aerobic/Anaerobic regulation system 2.1.1.1 Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3

Notebook : August 30

summary

• check the size by Gel electrophoresis for PCR clonies on Gibson assembly and ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .
• Do ligation for For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 , promoter fnr(activator)nirK + RBS_LacZ+Term_PSB3K3 and promoter fnr(repressor) + RBS_LacZ+Term_PSB3K3 and Transformation and

incubation.

lab work

• A.aero/anaerobic regulation system
  

1 -Gel electrophoresis of PCR clonies on Gibson assembly .

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Well 1 : 6µL DNA Ladder Well AA 1-8 : Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Well AB 1-8 : Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Concentrate3 Well AC 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Well AD 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Well AE 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Concentrate Well AF 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Concentrate Gel : 1.0%

2 -Gel electrophoresis of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .

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Well 0: 6µL DNA Ladder Well 1: clone1 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 Well 2: clone2 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 Well 3: clone3 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 Well 4: clone4 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 Gel : 1.0%

Ligation

For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3  :

promoter fnr(repressor)	8µl
RBS_LacZ+Term	3µl
Ligation buffer	2µl
Ligation T4 enzyme	2µl
H2O	6µl

Transformation theligation and spread in LB plates with Chlorenphenicol and Xgal. incubated at 37°C in aerobic condition over night.

For promoter fnr(activator)nirK + RBS_LacZ+Term_PSB3K3  :

promoter fnr(activator)nirK	3µl
RBS_LacZ+Term	3µl
Plasmid PSB3K3	5µl
Ligation buffer	2µl
Ligation T4 enzyme	1µl
H2O	6µl

Transformation theligation and spread in LB plates with k and Xgal. incubated at 37°C in anaerobic condition over night.

For promoter fnr(repressor) + RBS_LacZ+Term_PSB3K3  :

promoter fnr(repressor)	3µl
RBS_LacZ+Term	3µl
Plasmid PSB3K3	5µl
Ligation buffer	2µl
Ligation T4 enzyme	1µl
H2O	6µl

Transformation theligation and spread in LB plates with k and Xgal. incubated at 37°C in aerobic condition over night.

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