Team:Paris Saclay/Notebook/August/30

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(Created page with "='''Notebook : August 30'''= =='''summary'''== *Digetion of RBS_AmilCP+Term_PSB1C3 and RBS_LacZ+Term_PSB1C3 by SpeI and PstIand check the size by Gel electrophoresis * check...")
(→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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*Digetion of  RBS_AmilCP+Term_PSB1C3 and  RBS_LacZ+Term_PSB1C3 by SpeI and PstIand check the size by Gel electrophoresis
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==='''A - Aerobic/Anaerobic regulation system'''===
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* check the size by Gel electrophoresis for Degestion product of promoter fnr(activator)
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* We do PCR clonies on Gibson assembly and ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .
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* we got blue color of ligation promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in anaerobic condition over night.
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<br>
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=='''lab work'''==
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===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
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<br>
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===='''1 - Results of liquid culture of Pndh*-RBS-Amil CP-Term in pSB1C3 in aerobic and anaerobic conditions'''====
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*'''A.aero/anaerobic regulation system'''<br>
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 +
XiaoJing
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===='''1 -Gel electrophoresis of PCR clonies on Gibson assembly .'''====
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{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
IT WORKS !!!
 +
|}
 +
[[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
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
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.
 +
|}
 +
 +
[[File:PsPfnrcult3008.jpg|500px]]
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[[File:PsNirBcult3008.jpg|500px]]
 +
 +
===='''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 : [[Team:Paris_Saclay/Protocols/Transformation|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 : [[Team:Paris_Saclay/Protocols/Transformation|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
{|
{|
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| style="width:350px;border:1px solid black;" | [[]]
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| style="width:350px;border:1px solid black;" | [[File:Psgel13008.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
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*Well 1 : 6µL DNA Ladder
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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 2 : 20µL of Pndh* with RBS_AmilCP-Term clone 1 + 4µL of 6X loading dye
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*Well AB 1-8 : Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Concentrate3
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* Well 3 : 20µL of Pndh* with RBS_AmilCP-Term clone 2 + 4µL of 6X loading dye   
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*Well AC 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
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* Well 4 : 20µL of Pndh* with RBS_AmilCP-Term clone 3 + 4µL of 6X loading dye 
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*Well AD 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3
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* Well 5 : 20µL of Pndh* with RBS_AmilCP-Term clone 4 + 4µL of 6X loading dye 
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*Well AE 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Concentrate
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* Gel : 1.0%
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*Well AF 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Concentrate
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|}
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*Gel : 1.0%
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Expected sizes :
 +
* Pndh* with RBS_AmilCP-Term : 1000bp
 +
 
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size.  
|}
|}
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===='''2 -Gel electrophoresis of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .'''====
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===='''8 - Result of the purification colony of MG1655Z1 Δfnr'''====
 +
XiaoJing
 +
{|
 +
| 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.
 +
|}
 +
 +
[[File:PsLB3008.jpg|311px]][[File:PsKm3008.jpg|300px]]
 +
[[File:PsCm3008.jpg|311px]][[File:PsAm3008.jpg|311px]]
 +
 +
==='''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'''====
{|
{|
-
| style="width:350px;border:1px solid black;" | [[]]
+
| 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;" |
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*Well 0: 6µL DNA Ladder
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* Well 1 : 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 to 17 : 5µL of FNR + 1µl of 6X loading dye
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*Well 2: clone2 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
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* Well 18 to 26 : 5µL of RBS-BphR2 + 1µl of 6X loading dye
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*Well 3: clone3 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
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* Well 27 : 6µL DNA Ladder
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*Well 4: clone4 of ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
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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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*Gel : 1.0%
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* Well 50 : 6µL DNA Ladder
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* Gel : 1%
|}
|}
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Expect sizes :
 +
* FNR : 1096 bp
 +
* RBS-FNR : 1014 bp
 +
* RBS-BphR2 : 1469 bp
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good.
 +
|}
{| border="1" align="center"
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|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
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{{Team:Paris_Saclay/incl_fin}}
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Latest revision as of 01:23, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

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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