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
    - 1.1.1.1 Objective : obtaining Bba_K1155007
    - 1.1.1.2 1 - Extraction of Bba_K115007 from DH5α
  - 1.1.2 A - Aerobic/Anaerobic regulation system / B - PCB sensing system
    - 1.1.2.1 Objective : Obtaining FNR and BphR2 proteins
    - 1.1.2.2 2 - PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I

Notebook : August 9

Lab work

A - Aerobic/Anaerobic regulation system

((((((((((((((((((((====1 - Obtaining Δ fnr E. coli strain by transduction to test our biobricks====

Abdou, Anais, Damir, Nadia, XiaoJing

We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain was too old ( 2001)

Protocol : transduction

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.
We used a wild type strain to keep a stock and a Δ fnr E. coli strain to obtain phages that might have encapsidated a Δfnr::Km fragment.

10µl,50µl and 100µl petri dishes are clear so phages are multiplied.

We let the antibiotic over night to select the right strain. ))))))))))))))))))))))

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

CONCLUSION !!!!!!!! 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====

IMAGE

Well 1 : 6µL DNA Ladder
Well 2 : 5µL of BphR2 part1+1µl of 6X loading dy
Well 3 : 5µL of BphR2 part2+1µl of 6X loading dy
Well 4 : 5µL of RBS_BphR2 part1+1µl of 6X loading dy
Well 5 : 5µL of FNR part1+1µl of 6X loading dy
Well 6 : 5µL of FRN part2+1µl of 6X loading dy
Well 7: 5µL of RBS_FNR part1+1µl of 6X loading dy
Gel : 0.8%

we have no fragment so we must do again these PCRs )))))))))))))))))

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

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