Team:Paris Saclay/Notebook/August/9

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We observed lysis areas. We will continue the transduction protocol.
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We observed lysis areas in the strain MG1655Z1 Δfnr::Km after transduction. We will continue the transduction protocol.
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* 50µl phage: the petri dish is clear, bacteria are lysed by phages.  
* 50µl phage: the petri dish is clear, bacteria are lysed by phages.  
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Protocol : [[Team:Paris_Saclay/transduction|Transduction]]
 
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Our Mutant bacteria was called BW : Δfnr::Km.
 
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Our wild type bacteria was called MG1655Z1.
 
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We used kanamycine antibiotic.
 
===='''Objective : obtaining BBa_K1155007'''====
===='''Objective : obtaining BBa_K1155007'''====
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====1 - Extraction of BBa_K115007 from DH5α====
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====1 - Extraction of BBa_K115007 from DH5αstrain====
Abdou
Abdou
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Protocol : [[Team:Paris_Saclay/extraction|Hight copy plamid extraction]]
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Protocol : [[Team:Paris_Saclay/extraction|High-copy plamid extraction]]
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We used colonies number 10, 14 and 15.
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We extracted plamid from colonies number 10, 14 and 15.
Nanodrop
Nanodrop
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The extraction was good. We will sequence our plasmid.  
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The extraction was good. We will sequence our plasmids.  
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*Well 1 : 6µL DNA Ladder
*Well 1 : 6µL DNA Ladder
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*Well 2 : 5µL of BphR2 Part I+1µl of 6X loading dye
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*Well 2 : 5µL of BphR2 Part I + 1µl of 6X loading dye
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*Well 3 : 5µL of BphR2 Part II+1µl of 6X loading dye
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*Well 3 : 5µL of BphR2 Part II + 1µl of 6X loading dye
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*Well 4 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye
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*Well 4 : 5µL of RBS-BphR2 Part I + 1µl of 6X loading dye
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*Well 5 : 5µL of FNR Part I+1µl of 6X loading dye
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*Well 5 : 5µL of FNR Part I + 1µl of 6X loading dye
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*Well 6 : 5µL of FRN Part II+1µl of 6X loading dye
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*Well 6 : 5µL of FRN Part II + 1µl of 6X loading dye
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*Well 7: 5µL of RBS-FNR Part I+1µl of 6X loading dye
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*Well 7: 5µL of RBS-FNR Part I + 1µl of 6X loading dye
*Gel : 0.8%
*Gel : 0.8%
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Latest revision as of 15:28, 4 October 2013

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Contents

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, XiaoJing

We observed lysis areas in the strain MG1655Z1 Δfnr::Km after transduction. We will continue the transduction protocol.

Ps908transduction.jpg

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.

Objective : obtaining BBa_K1155007

1 - Extraction of BBa_K115007 from DH5αstrain

Abdou

Protocol : High-copy plamid extraction

We extracted plamid from 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 plasmids.

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

Psgel10908.jpg
  • 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 :

PsPCRBphR23007.jpg

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

PsPCRFNR3007.jpg


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