Team:Paris Saclay/Notebook/August/28

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : characterize Bba_K1155000 and Bba_K1155004'''====
+
===='''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'''====
+
===='''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
XiaoJing
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DE;" |
| 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.   
+
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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We streak :  
+
We streak colonies from construction :  
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* Pfnr with RBS-LacZ-Term in PSB1C3 with O2 and Xgal
+
* Pndh* with RBS-LacZ-Term in pSB1C3 with O2 and Xgal
-
* Pfnr with RBS-Amil CP-Term in PSB1C3 with O2  
+
* Pndh* with RBS-Amil CP-Term in pSB1C3 with O2  
-
* Pfnr with RBS-Amil CP-Term in PSB1C3 without 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-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 with O2
-
* NirB with RBS-Amil CP-Term in PSB1C3 without O2
+
* NirB with RBS-Amil CP-Term in pSB1C3 without O2
 +
===='''2 - Culture of mutant strain MG1655Z1 Δfnr''' ====
 +
 +
XiaoJing
 +
 +
{|
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
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'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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===='''Objective : obtaining FRN and BphR2 proteins'''====
===='''Objective : obtaining FRN and BphR2 proteins'''====
-
===='''1 - Gel purification of PSB1C3 digested by DnpI '''====
+
===='''1 - Gel purification of pSB1C3 digested by DnpI '''====
XiaoJing
XiaoJing
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Nanodrop :  
Nanodrop :  
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* PSB1C3 : 37.2ng/µL
+
* pSB1C3 : 37.2ng/µL
-
===='''2 - Electrophoresis to check the gel purification of PSB1C3 digested by DnpI'''====
+
===='''2 - Electrophoresis to check the gel purification of pSB1C3 digested by DnpI'''====
XiaoJing  
XiaoJing  
{|
{|
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| style="width:350px;border:1px solid black;" |]]
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| style="width:350px;border:1px solid black;" |[[File:Psgel12808.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 : 3µL of PSB1C3 digested by DpnI+1µl of 6X loading dye
+
* Well 2 : 3µL of pSB1C3 digested by DpnI + 1µl of 6X loading dye
* Gel : 1%
* Gel : 1%
|}
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expected sizes :  
expected sizes :  
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* PSB1C3 : 2070 bp
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* pSB1C3 : 2070 bp
{|
{|
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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We obtain fragment at the right size. The gel purification was good. We will use it for Gibson assembly.
+
We obtained a fragment at the right size. The gel purification was good. We will use it for Gibson assembly.
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* RBS-BphR2 :  
* RBS-BphR2 :  
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** PSB1C3 : 3µL
+
** pSB1C3 : 3µL
** BphR2 Part I : 1µL  
** BphR2 Part I : 1µL  
** BphR2 Part II : 1µL  
** BphR2 Part II : 1µL  
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* FNR :  
* FNR :  
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** PSB1C3 : 3µL
+
** pSB1C3 : 3µL
** FNR Part I : 1µL
** FNR Part I : 1µL
** FNR Part II : 1µL
** FNR Part II : 1µL
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* RBS-FNR :
* RBS-FNR :
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** PSB1C3 : 3µL
+
** pSB1C3 : 3µL
** RBS-FNR Part I : 1µL
** RBS-FNR Part I : 1µL
** FNR Part II : 1µL
** FNR Part II : 1µL
** Gibson mix : 15µL  
** Gibson mix : 15µL  
-
We keep these mix at 50°C for 1h.
+
We incubate these mix at 50°C during 1h inside PCR machine.
-
===='''2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α'''====
+
===='''2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α strain'''====
XiaoJing  
XiaoJing  

Latest revision as of 01:20, 5 October 2013

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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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