Team:Paris Saclay/Notebook/August/30

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(→1 - Electrophoresis of PCR Colony of FNR, RBS-FNR and RBS-BphR2)
 
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{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : August 30'''=
='''Notebook : August 30'''=
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=='''summary'''==
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=='''Lab work'''==
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* check the size by Gel electrophoresis for  PCR clonies on Gibson assembly and ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .
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* Do ligation for For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 , promoter fnr(activator)nirK + RBS_LacZ+Term_PSB3K3 and promoter fnr(repressor) + RBS_LacZ+Term_PSB3K3 and Transformation and
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incubation.
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<br>
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=='''lab work'''==
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<br>
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*'''A.aero/anaerobic regulation system'''<br>
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===='''1 -Gel electrophoresis of PCR clonies on Gibson assembly .'''====
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 +
==='''A - Aerobic/Anaerobic regulation system'''===
 +
===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
 +
===='''1 - Results of liquid culture of Pndh*-RBS-Amil CP-Term in pSB1C3 in aerobic and anaerobic conditions'''====
 +
XiaoJing
{|
{|
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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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IT WORKS !!!
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*Well 1 : 6µL DNA Ladder
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*Well AA 1-8 : Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3
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*Well AB 1-8 : Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Concentrate3
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*Well AC 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
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*Well AD 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3
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*Well AE 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Concentrate
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*Well AF 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Concentrate
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*Gel : 1.0%
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|}
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===='''2 -Gel electrophoresis of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .'''====
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[[File:PsPfnr3008.jpg|600px]]
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In anaerobic condition, FNR protein (active form) binds to the constitutive promoter Pndh* and repressed ''amilCP'' expression. Therefore, the bacteria have no colour.
 +
In aerobic condition, FNR protein is inactive and can not bind to constitutive promoter Pndh*, ''amilCP'' is expressed and we can see the bacteria have violet colour.
 +
===='''2 - Results of culture of Pndh* with RBS-AmilCP-Term in pSB1C3, PnirB with RBS-LacZ-Term in pSB1C3 in aerobic and anaerobic conditions'''====
 +
XiaoJing
{|
{|
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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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Purification of 08/29/13 didn't work. We have blue colonies for Pndh* with RBS-AmilCP-Term in pSB1C3 in aerobic and anaerobic conditions. We also have blue colonies for PnirB with RBS-LacZ-Term in pSB1C3 in anaerobic conditions.
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*Well 0: 6µL DNA Ladder
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*Well 1: clone1 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
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*Well 2: clone2 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
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*Well 3: clone3 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
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*Well 4: clone4 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
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*Gel : 1.0%
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|}
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[[File:PsPfnrcult3008.jpg|500px]]
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[[File:PsNirBcult3008.jpg|500px]]
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===='''3 - Ligation of Pndh* with RBS-LacZ-Term in pSB1C3 '''====
 +
XiaoJing
 +
Used quantities :
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* Pndh* : 8µL
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* RBS-LacZ-Term in pSB1C3 : 3µL
 +
* Buffer ligation : 2µL
 +
* Ligase : 1µL
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* H2O : 6µL
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:<u>Ligation</u>
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===='''4 - Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB1C3 in DH5α strain '''====
 +
XiaoJing
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Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
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:For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 :
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We incubate the bacteria in LB-chloramphenicol-Xgal plate at 37°C in aerobic conditions.
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{| border="1" align="center"
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===='''5 - Ligation of Pfnr with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3 '''====
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|-
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|promoter fnr(repressor)
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|8µl
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|-
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|RBS_LacZ+Term
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|3µl
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|-
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|Ligation buffer
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|2µl
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|-
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|Ligation T4 enzyme
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|2µl
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|-
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|H2O
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|6µl
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|}
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Transformation theligation and spread in LB plates with Chlorenphenicol and Xgal.
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XiaoJing
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incubated at 37°C in aerobic condition over night.
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:For promoter fnr(activator)nirK + RBS_LacZ+Term_PSB3K3  :
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Used quantities :  
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* Pndh*, PnarK : 3µL
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* RBS-LacZ-Term : 3µL
 +
* pSB3K3 : 5µL
 +
* Buffer ligase : 2µL
 +
* Ligase : 1µL
 +
* H2O : 6µL
 +
===='''6 - Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3 in DH5α strain '''====
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{| border="1" align="center"
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XiaoJing
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|-
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|promoter fnr(activator)nirK
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|3µl
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|-
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|RBS_LacZ+Term
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|3µl
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|-
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|Plasmid PSB3K3
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|5µl
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|-
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|Ligation buffer
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|2µl
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|-
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|Ligation T4 enzyme
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|1µl
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|-
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|H2O
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|6µl
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|}
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Transformation theligation and spread in LB plates with k and Xgal.
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Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
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incubated at 37°C in anaerobic condition over night.
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We incubate the bacteria at 37°C with LB-Kanamycine-Xgal in anaerobic conditions for PnarK with RBS-LacZ-Term in pSB3K3.
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:For promoter fnr(repressor) + RBS_LacZ+Term_PSB3K3  :
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We incubate the bacteria at 37°C with LB-Kanamycine-Xgal in aerobic conditions for Pndh* with RBS-LacZ-Term in pSB3K3.
 +
===='''7 - Electrophoresis of  PCR Colony of Pndh* with RBS_AmilCP-Term  in DH5α strain '''====
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{| border="1" align="center"
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XiaoJing
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|-
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|promoter fnr(repressor)
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{|
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|3µl
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| style="width:350px;border:1px solid black;" | [[File:Psgel13008.jpg]]
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|-
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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|RBS_LacZ+Term
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* Well 1 : 6µL DNA Ladder
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|3µl
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* Well 2 : 20µL of Pndh* with RBS_AmilCP-Term clone 1 + 4µL of 6X loading dye
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|-
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* Well 3 : 20µL of Pndh* with RBS_AmilCP-Term clone 2 + 4µL of 6X loading dye   
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|Plasmid PSB3K3
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* Well 4 : 20µL of Pndh* with RBS_AmilCP-Term clone 3 + 4µL of 6X loading dye 
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|5µl
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* Well 5 : 20µL of Pndh* with RBS_AmilCP-Term clone 4 + 4µL of 6X loading dye 
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|-
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* Gel : 1.0%
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|Ligation buffer
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|2µl
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|-
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|Ligation T4 enzyme
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|1µl
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|-
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|H2O
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|6µl
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|}
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Transformation theligation and spread in LB plates with k and Xgal.
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Expected sizes :
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incubated at 37°C in aerobic condition over night.
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* Pndh* with RBS_AmilCP-Term : 1000bp
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{|
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{| border="1" align="center"
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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||<big>Previous week</big>
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We obtained fragments at the right size.
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|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
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|[[Team:Paris Saclay/Notebook/August/30|<big>Next day</big>]]
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|}
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===='''8 - Result of the purification colony of MG1655Z1 Δfnr'''====
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{{Team:Paris_Saclay/incl_fin}}
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{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : August 23'''=
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=='''Lab work'''==
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-
 
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==='''A - Aerobic/Anaerobic regulation system'''===
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-
 
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-
===='''Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+
-
 
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-
===='''1 - Results of liquid culture of Pfnr-RBS-Amil CP-Term in PSB1C3 in aerobic and anaerobic conditions'''====
+
XiaoJing
XiaoJing
-
IT WORKS !!!
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
It works. Our plasmid didn't contain any antibiotic resistance gene. Colonies grew in LB medium and didn't grow on kanamycin, ampicilin, chloramphenicol medium. We obtain strain MG1655Z1 Δfnr.
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|}
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[[File:PsPfnr3008.jpg|600px]]
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[[File:PsLB3008.jpg|311px]][[File:PsKm3008.jpg|300px]]
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[[File:PsCm3008.jpg|311px]][[File:PsAm3008.jpg|311px]]
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In anaerobic conditions, thank to promotor RBS-Amil CP-Term, the dectetion gene is activated and we can see a violet coloration of bacterias.  
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In aerobic conditions, promotor RBS-Amil CP-Term is inactive, the dectetion gene isn't activated and we can't see any coloration of bacterias.  
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===='''1 - Ligation of Pfnr with RBS-LacZ-Term in PSB1C3 '''====
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-
 
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-
XiaoJing
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-
 
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Used quantities :  
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* Pfnr : 8µL
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* RBS-LacZ-Term in PSB1C3 : 3µL
+
-
* Buffer ligation : 2µL
+
-
* Ligase : 1µL
+
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* H2O : 6µL
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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===='''Objective : obtaining FNR and BphR2 proteins'''====
===='''Objective : obtaining FNR and BphR2 proteins'''====
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===='''1 - Electrophoresis of Colony PCR of FNR, RBS-FNR and RBS-BphR2'''====
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===='''1 - Electrophoresis of PCR Colony of FNR, RBS-FNR and RBS-BphR2'''====
{|
{|
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| style="width:350px;border:1px solid black;" |]]
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| style="width:350px;border:1px solid black;" |[[File:Psgel23008.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL DNA Ladder
* Well 1 : 6µL DNA Ladder
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* Well 2 to 17 : 5µL of FNR+1µl of 6X loading dye
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* Well 2 to 17 : 5µL of FNR + 1µl of 6X loading dye
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* Well 18 to 26 : 5µL of RBS-BphR2+1µl of 6X loading dye
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* Well 18 to 26 : 5µL of RBS-BphR2 + 1µl of 6X loading dye
* Well 27 : 6µL DNA Ladder
* Well 27 : 6µL DNA Ladder
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* Well 28 to 41 : 5µL of RBS-FNR+1µl of 6X loading dye
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* Well 28 to 41 : 5µL of RBS-FNR + 1µl of 6X loading dye
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* Well 42 to 49 : 5µL of RBS-BphR2+1µl of 6X loading dye
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* Well 42 to 49 : 5µL of RBS-BphR2 + 1µl of 6X loading dye
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* WEll 50 : 6µL DNA Ladder
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* Well 50 : 6µL DNA Ladder
* Gel : 1%
* Gel : 1%
|}
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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We obtain fragment at right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good.
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We obtained fragments at the right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good.
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|}
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{| border="1" align="center"
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|[[Team:Paris Saclay/Notebook/August/29|<big>Previous day</big>]]
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|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
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|[[Team:Paris Saclay/Notebook/August/31|<big>Next day</big>]]
|}
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 01:23, 5 October 2013

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Contents

Notebook : August 30

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Results of liquid culture of Pndh*-RBS-Amil CP-Term in pSB1C3 in aerobic and anaerobic conditions

XiaoJing

IT WORKS !!!

PsPfnr3008.jpg

In anaerobic condition, FNR protein (active form) binds to the constitutive promoter Pndh* and repressed amilCP expression. Therefore, the bacteria have no colour.

In aerobic condition, FNR protein is inactive and can not bind to constitutive promoter Pndh*, amilCP is expressed and we can see the bacteria have violet colour.

2 - Results of culture of Pndh* with RBS-AmilCP-Term in pSB1C3, PnirB with RBS-LacZ-Term in pSB1C3 in aerobic and anaerobic conditions

XiaoJing

Purification of 08/29/13 didn't work. We have blue colonies for Pndh* with RBS-AmilCP-Term in pSB1C3 in aerobic and anaerobic conditions. We also have blue colonies for PnirB with RBS-LacZ-Term in pSB1C3 in anaerobic conditions.

PsPfnrcult3008.jpg PsNirBcult3008.jpg

3 - Ligation of Pndh* with RBS-LacZ-Term in pSB1C3

XiaoJing

Used quantities :

  • Pndh* : 8µL
  • RBS-LacZ-Term in pSB1C3 : 3µL
  • Buffer ligation : 2µL
  • Ligase : 1µL
  • H2O : 6µL

4 - Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB1C3 in DH5α strain

XiaoJing

Protocol : Bacterial transformation

We incubate the bacteria in LB-chloramphenicol-Xgal plate at 37°C in aerobic conditions.

5 - Ligation of Pfnr with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3

XiaoJing

Used quantities :

  • Pndh*, PnarK : 3µL
  • RBS-LacZ-Term : 3µL
  • pSB3K3 : 5µL
  • Buffer ligase : 2µL
  • Ligase : 1µL
  • H2O : 6µL

6 - Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3 in DH5α strain

XiaoJing

Protocol : Bacterial transformation

We incubate the bacteria at 37°C with LB-Kanamycine-Xgal in anaerobic conditions for PnarK with RBS-LacZ-Term in pSB3K3.

We incubate the bacteria at 37°C with LB-Kanamycine-Xgal in aerobic conditions for Pndh* with RBS-LacZ-Term in pSB3K3.

7 - Electrophoresis of PCR Colony of Pndh* with RBS_AmilCP-Term in DH5α strain

XiaoJing

Psgel13008.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 20µL of Pndh* with RBS_AmilCP-Term clone 1 + 4µL of 6X loading dye
  • Well 3 : 20µL of Pndh* with RBS_AmilCP-Term clone 2 + 4µL of 6X loading dye
  • Well 4 : 20µL of Pndh* with RBS_AmilCP-Term clone 3 + 4µL of 6X loading dye
  • Well 5 : 20µL of Pndh* with RBS_AmilCP-Term clone 4 + 4µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • Pndh* with RBS_AmilCP-Term : 1000bp

We obtained fragments at the right size.

8 - Result of the purification colony of MG1655Z1 Δfnr

XiaoJing

It works. Our plasmid didn't contain any antibiotic resistance gene. Colonies grew in LB medium and didn't grow on kanamycin, ampicilin, chloramphenicol medium. We obtain strain MG1655Z1 Δfnr.

PsLB3008.jpgPsKm3008.jpg PsCm3008.jpgPsAm3008.jpg

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

Objective : obtaining FNR and BphR2 proteins

1 - Electrophoresis of PCR Colony of FNR, RBS-FNR and RBS-BphR2

Psgel23008.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 to 17 : 5µL of FNR + 1µl of 6X loading dye
  • Well 18 to 26 : 5µL of RBS-BphR2 + 1µl of 6X loading dye
  • Well 27 : 6µL DNA Ladder
  • Well 28 to 41 : 5µL of RBS-FNR + 1µl of 6X loading dye
  • Well 42 to 49 : 5µL of RBS-BphR2 + 1µl of 6X loading dye
  • Well 50 : 6µL DNA Ladder
  • Gel : 1%

Expect sizes :

  • FNR : 1096 bp
  • RBS-FNR : 1014 bp
  • RBS-BphR2 : 1469 bp

We obtained fragments at the right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good.


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