Team:Paris Saclay/Notebook/August/28

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='''Notebook : August 28'''=
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=='''Lab work'''==
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==='''A - Aerobic/Anaerobic regulation system'''===
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===='''Objective : characterize Bba_K1155000 and Bba_K1155004'''====
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===='''1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions'''====
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XiaoJing
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{|
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
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Transformation of 08/26/13 works. We will use it to characterize all ligations. PCR colony of 08/27/13 works for . We also will use them to characterize ligations : NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3. 
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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===='''Objective : obtaining FRN and BphR2 proteins'''====
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===='''1 - Gel purification of PSB1C3 digested by DnpI '''====
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XiaoJing
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Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
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Nanodrop :
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* PSB1C3 : 37.2ng/µL
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===='''2 - Electrophoresis to check the gel purification of PSB1C3 digested by DnpI'''====
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XiaoJing
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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 : 3µL of PSB1C3 digested by DpnI+1µl of 6X loading dye
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* Gel : 1%
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expected sizes :
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* PSB1C3 : 2070 bp
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{|
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
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We obtain frragment at the right size. The gel purification was good. We will use it for Gibson assembly.
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|}
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===='''3 - Gibson assembly'''====
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XiaoJing
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Used quantities :
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* RBS-BphR2 :
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** PSB1C3 : 3µL
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** BphR2 Part I : 1µL
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** BphR2 Part II : 1µL
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** Gibson mix : 15µL ???????????????
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* FNR :
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** PSB1C3 : 3µL
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** FNR Part I : 1µL
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** FNR Part II : 1µL
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** Gisbon mix : 15µL ???????????????
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* RBS-FNR :
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** PSB1C3 : 3µL
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** RBS-FNR Part I : 1µL
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** FNR Part II : 1µL
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** Gibson mix : 15µL ????????????
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We let these mix at 50°C during 1h.
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 21:42, 21 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 28

summary

  • We got purple clonies on ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3

so we do a purify of 4 clonies in two plate.incubated at 37°C in aerobic and anaerobic condition over night to verify the different.

  • Prepare LB plates with Chlorenphenicol and Xgal .Pufify single conle of promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 and promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3 to see if color will change.
  • Gel extration of Digestion Dnp1 purification of the the PSB1C3 clean for Gibson and do Gibson assembly again .


lab work


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

  • 1 - Gel extraction of the PSB1C3 Clean for Gibson.

Protocol : Gel extraction

  • 2- Migration of 3µl PSB1C3 Clean for Gibson (product gel extraction) '
[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of PSB1C3 Clean dig Dnp1 gel extraction


  • Gel : 1.0%
  • 3- product quantification.
We used the nano drop to measure the DNA in 260nm and we found its concentration
name PSB1C3 Clean dig Dnp1 gel extraction
conc. 37.2ng/µl



  • 4 - Gibson assembly.


  • 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 : RBS_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.



A - Aerobic/Anaerobic regulation system

  • 1 - Purify single clone of promoter fnr(activator)nirB + RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in anaerobic condition over night.

Protocol : Purify single clone

  • 2 - Purify single clone of promoter promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in aerobic condition over night
  • 3 - Purify single clone of promoter fnr(activator)nirB + RBS_AmilCP+Term_PSB1C3 on plates wiht Chlorenphenicol.incubated at 37°C in anaerobic condition over night


  • 4 - Purify 4 single purple colony of promoter fnr(repressor)+ RBS_AmilCP+Term_PSB1C3 on two plates wiht Chlorenphenicol .incubated at 37°C in aerobic and anaerobic condition over night


  • 5- Purify single clone of B5 promoter fnr(activator)nirB + RBS_AmilCP+Term_PSB1C3 on plates wiht Chlorenphenicol.incubated at 37°C in anaerobic condition over night


  • 6- Purify single clone of D1, D5, D6 promoter fnr(repressor)+ RBS_AmilCP+Term_PSB1C3 on two plates wiht Chlorenphenicol .incubated at 37°C in aerobic and anaerobic condition over night


  • 7- Purify single clone of C1, C2, C5 ,C6promoter promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in aerobic condition over night


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Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 28

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000 and Bba_K1155004

1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions

XiaoJing

Transformation of 08/26/13 works. We will use it to characterize all ligations. PCR colony of 08/27/13 works for . We also will use them to characterize ligations : NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3.

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

Objective : obtaining FRN and BphR2 proteins

1 - Gel purification of PSB1C3 digested by DnpI

XiaoJing

Protocol : Gel purification

Nanodrop :

  • PSB1C3 : 37.2ng/µL

2 - Electrophoresis to check the gel purification of PSB1C3 digested by DnpI

XiaoJing

]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of PSB1C3 digested by DpnI+1µl of 6X loading dye
  • Gel : 1%

expected sizes :

  • PSB1C3 : 2070 bp

We obtain frragment at the right size. The gel purification was good. We will use it for Gibson assembly.

3 - Gibson assembly

XiaoJing

Used quantities :

  • RBS-BphR2 :
    • PSB1C3 : 3µL
    • BphR2 Part I : 1µL
    • BphR2 Part II : 1µL
    • Gibson mix : 15µL ???????????????
  • FNR :
    • PSB1C3 : 3µL
    • FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gisbon mix : 15µL ???????????????
  • RBS-FNR :
    • PSB1C3 : 3µL
    • RBS-FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gibson mix : 15µL ????????????

We let these mix at 50°C during 1h.

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