Team:Paris Saclay/Notebook/August/9

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Contents

1 Notebook : August 9
- 1.1 Lab work
  - 1.1.1 A - Aerobic/Anaerobic regulation system
  - 1.1.2 A - Aerobic/Anaerobic regulation system / B - PCB sensing system

Notebook : August 9

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Tranduction of Km in MG1655Z1

Abdou, Anaïs, Damir, Nadia, XioaJing

We observed lysis areas. We will continue the transduction protocol.

Picture: lysed cells comparison.

0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
50µl phage: the petri dish is clear, bacteria are lysed by phages.

Protocol : Transduction

Our Mutant bacteria was called BW : Δfnr::Km. Our wild type bacteria was called MG1655Z1. We used kanamycine antibiotic.

Objective : obtaining BBa_K1155007

1 - Extraction of BBa_K115007 from DH5α

Abdou

Protocol : Hight copy plamid extraction

We used colonies number 10, 14 and 15.

Nanodrop

BBa_K1155007 in clone 10 : 38ng/µl
BBa_K1155007 in clone 14 : 48.5ng/µl
BBa_K1155007 in clone 15 : 52 ng/µl

The extraction was good. We will sequence our plasmid.

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

Objective : Obtaining FNR and BphR2 proteins

1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification

IMAGE

Well 1 : 6µL DNA Ladder
Well 2 : 5µL of BphR2 Part I+1µl of 6X loading dye
Well 3 : 5µL of BphR2 Part II+1µl of 6X loading dye
Well 4 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye
Well 5 : 5µL of FNR Part I+1µl of 6X loading dye
Well 6 : 5µL of FRN Part II+1µl of 6X loading dye
Well 7: 5µL of RBS-FNR Part I+1µl of 6X loading dye
Gel : 0.8%

Expected size :

BphR2 Part I : 178 bp
BphR2 Part II : 790bp
RBS-BphR2 Part I : 197bp
FNR Part I : 597 bp
FNR Part II : 200bp
RBS-FNR PartI : 615bp

We lost all our PCR fragments. We will do the PCR again.

2 - PCR of BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I

Anaïs, Damir, Nadia, XiaoJing

Used quantities :

Bphr2 Part I :
- Oligo 54F : 1µL
- Oligo 55R : 1µL
- Buffer Phusion : 10µL
- DNA of Pseudomonas pseudoalcaligenes : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

Bphr2 Part II :
- Oligo 56F : 1µL
- Oligo 57R : 1µL
- Buffer Phusion : 10µL
- DNA Pseudomonas pseudoalcaligenes : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

RBS-Bphr2 Part I :
- Oligo 58F : 1µL
- Oligo 57R : 1µL
- Buffer Phusion : 10µL
- DNA Pseudomonas pseudoalcaligenes : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

FNR Part I :
- Oligo 59F : 1µL
- Oligo 60R : 1µL
- Buffer Phusion : 10µL
- DNA Escherichia coli : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

FNR Part II :
- Oligo 61F : 1µL
- Oligo 62R : 1µL
- Buffer Phusion : 10µL
- DNA Escherichia coli : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

RBS-FNR Part I :
- Oligo 63F : 1µL
- Oligo 62R : 1µL
- Buffer Phusion : 10µL
- DNA Escherichia coli : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

PCR Program :

BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :

FNR Part I, FNR Part II, RBS-FNR Part I :

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