Team:Paris Saclay/Notebook/August/21

From 2013.igem.org

(Difference between revisions)
(→Obtaining FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 or RBS_Amicip+Term_PSB1C3)
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Incubate at 37°C over night with shake 180 rpm.
Incubate at 37°C over night with shake 180 rpm.
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====4 - Extract plasmid from strain BBa_K115500 clone1 by kit ====
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===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''====
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obtain plasmid 50µL concentration 195ng/µL
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====1 - Electrophoresis of the digestion of Bba_J04450 by EcoRI/PstI====
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XiaoJing
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=====5 - Digestion of Bba_K1155000 by SpeI =====
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{|
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| style="width:350px;border:1px solid black;" | [[]]
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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* Well 1 : 6µL DNA Ladder
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* Well 2 : 17µL of Bba_J04450 digested by EcoRI/PstI+3µL of H2O+4µL of 6X loading buffer
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* Gel : 1%
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|}
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Expected sizes :
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* PSB3K3 : 2750 bp
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* GFP : ... NON VISIBLE SUR LE GEL ???????
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* Digestion by SpeI :
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{|
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**Buffer : 2µL
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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**SpeI : 2µL
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We obtain a fragment at the right size. We can purify it.
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**Plasmid : 10µL
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**H20 : 6µL
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Do 3 tubes, Incubate at 37°C for 2 hours
 
==='''A - Aerobic/Anaerobic regulation system / B - PCB se nsing system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB se nsing system'''===
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These 3 mixture is incubated at 50°C for up to one hour.
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.
Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.
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Revision as of 22:16, 11 September 2013

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Contents

Notebook : August 21

Lab work

A - Aerobic/Anaerobic regulation system

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

1 - Colony PCR on e.coli with FNR Promoter (nirB,narG,narK)plus RBS_LacZ+Term_PSB1C3 or RBS_AmilCP+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_AmilCP+Term_PSB1C3
  • C1-8: FNR Promoter narG plus RBS_LacZ+Term_PSB1C3
  • D1-8: FNR Promoter narG plus RBS_AmilCP+Term_PSB1C3
  • E1-8: FNR Promoter narK plus RBS_LacZ+Term_PSB1C3
  • F1-8: FNR Promoter narK Plus RBS_AmilCP+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




2 - Gel electrophoresis of the colony PCR products
[[]]
  • 6µL DNA Ladder
  • 10µL sample per well
  • Gel : 0.8%

Expected size : 3583bp

3 - Culture

Nguyen

  • BBa_K115500 strains (FNR repressor)in 5ml LB with Chlorenphenicol
  • strain MG1665 Δ fnr:km clone1 in 5ml LB with kanamicine

Incubate at 37°C over night with shake 180 rpm.

Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3

1 - Electrophoresis of the digestion of Bba_J04450 by EcoRI/PstI

XiaoJing

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 17µL of Bba_J04450 digested by EcoRI/PstI+3µL of H2O+4µL of 6X loading buffer
  • Gel : 1%

Expected sizes :

  • PSB3K3 : 2750 bp
  • GFP : ... NON VISIBLE SUR LE GEL ???????

We obtain a fragment at the right size. We can purify it.


A - Aerobic/Anaerobic regulation system / B - PCB se nsing system

1 - Gibson assembloy.

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.




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