Team:Paris Saclay/Notebook/August/28

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='''Notebook : August 28'''=
='''Notebook : August 28'''=
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=='''summary'''==  
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=='''Lab work'''==
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*We got purple clonies on ligation promoter fnr(repressor) plus  RBS_AmilCP+Term_PSB1C3
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==='''A - Aerobic/Anaerobic regulation system'''===
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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.
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*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.
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* Gel extration of Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson and do Gibson assembly again .
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===='''Objective : characterize BBa_K1155000 and BBa_K1155004'''====
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<br>
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=='''lab work'''==
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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, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions'''====
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<br>
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XiaoJing
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'''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''
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*''1 - Gel extraction of the PSB1C3 Clean for Gibson.''
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Protocol : [[Team:Paris_Saclay/Protocols/Gel extraction|Gel extraction]]
 
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*''2- Migration of 3µl PSB1C3 Clean for Gibson (product gel extraction) '
 
{|
{|
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| style="width:350px;border:1px solid black;" | [[]]
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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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, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3. 
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*Well 1 : 6µL DNA Ladder
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|}
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*Well 2 : 3µL of PSB1C3 Clean dig Dnp1 gel extraction
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We streak colonies from construction :
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* Pndh* with RBS-LacZ-Term in pSB1C3 with O2 and Xgal
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* Pndh* with RBS-Amil CP-Term in pSB1C3 with O2
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* Pndh* with RBS-Amil CP-Term in pSB1C3 without O2
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* NirB with RBS-LacZ-Term in pSB1C3 without O2 and Xgal
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* NirB with RBS-Amil CP-Term in pSB1C3 with O2
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* NirB with RBS-Amil CP-Term in pSB1C3 without O2
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*Gel : 1.0%
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===='''2 - Culture of mutant strain MG1655Z1 Δfnr''' ====
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|}
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*''3- product quantification.''
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:We used the nano drop to measure the DNA in 260nm and we found its concentration
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{| border="1" align="center"
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|-
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|name
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| PSB1C3 Clean dig Dnp1 gel extraction
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|-
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XiaoJing
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|conc.
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|37.2ng/µl
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{|
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
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Transformation of 08/27/13 works. We will do a purification on LB plates.
|}
|}
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<br>
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We streaked our colonies on plates with LB and incubated them at 42°C.
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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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*''4 - Gibson assembly.''
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===='''1 - Gel purification of pSB1C3 digested by DnpI '''====
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XiaoJing
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* RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
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Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
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* FNR_part1, FNR part1 and plasmid PSB1C3
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* RBS_FNR part1, FNR_part2 and plasmid PSB1C3
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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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Sample Volume:
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XiaoJing
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{|
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| style="width:700px;border:1px solid black;vertical-align:top;" |
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{|
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*Tube 1 : RBS_BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
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| style="width:350px;border:1px solid black;" |[[File:Psgel12808.jpg]]
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*Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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*Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
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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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These 3 mixture is incubated at 50°C for up to one hour.
 
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Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.
 
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expected sizes :
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* pSB1C3 : 2070 bp
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<br>
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{|
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'''A - Aerobic/Anaerobic regulation system '''
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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We obtained a fragment at the right size. The gel purification was good. We will use it for Gibson assembly.
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|}
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*''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. ''
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===='''3 - Gibson assembly'''====
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Protocol : [[Team:Paris_Saclay/Protocols/Purify single clone|Purify single clone]]
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*''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''
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XiaoJing
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*''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''
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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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*''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''
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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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*''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''
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We incubate these mix at 50°C during 1h inside PCR machine.
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===='''2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α strain'''====
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*''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''
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XiaoJing
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Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
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*''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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||<big>Previous week</big>
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|[[Team:Paris Saclay/Notebook/August/29|<big>Next day</big>]]
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{{Team:Paris_Saclay/incl_fin}}
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Latest revision as of 01:20, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

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, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* 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, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3.

We streak colonies from construction :

  • Pndh* with RBS-LacZ-Term in pSB1C3 with O2 and Xgal
  • Pndh* with RBS-Amil CP-Term in pSB1C3 with O2
  • Pndh* with RBS-Amil CP-Term in pSB1C3 without O2
  • NirB with RBS-LacZ-Term in pSB1C3 without O2 and Xgal
  • NirB with RBS-Amil CP-Term in pSB1C3 with O2
  • NirB with RBS-Amil CP-Term in pSB1C3 without O2

2 - Culture of mutant strain MG1655Z1 Δfnr

XiaoJing

Transformation of 08/27/13 works. We will do a purification on LB plates.

We streaked our colonies on plates with LB and incubated them at 42°C.

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

Psgel12808.jpg
  • 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 obtained a fragment 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 incubate these mix at 50°C during 1h inside PCR machine.

2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α strain

XiaoJing

Protocol : Bacterial transformation


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