Team:Paris Saclay/Notebook/August/21

From 2013.igem.org

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+
===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
=====1 - Colony PCR of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α=====
+
====1 - Colony PCR of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α====
Damir, Xiaojing
Damir, Xiaojing
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
The transformation was good. We will do a Colony PCR.
 +
|}
Colonies repiquée dans 10µL d'eau.
Colonies repiquée dans 10µL d'eau.
Line 23: Line 28:
** H2O : 1.88mL
** H2O : 1.88mL
-
* NirB with RBS-LacZ-Term in PSB1C3 :  
+
*** NirB with RBS-LacZ-Term in pSB1C3 :  
-
** 45µL of Mix A+1µL of oligo 45+2µL of DNA
+
**** 45µL of Mix A+1µL of oligo 45+2µL of DNA
-
* NirB with RBS-Amil CP-Term in PSB1C3 :  
+
*** NirB with RBS-Amil CP-Term in pSB1C3 :  
-
** 45µL of Mix A+1µL of oligo 45 +2µL of DNA
+
**** 45µL of Mix A+1µL of oligo 45 +2µL of DNA
-
* NarG with RBS-LacZ-Term in PSB1C3 :  
+
*** NarG with RBS-LacZ-Term in pSB1C3 :  
-
** 45µL of Mix A+1µL of oligo 41+2µL of DNA
+
**** 45µL of Mix A+1µL of oligo 41+2µL of DNA
-
* NarG with RBS-Amil CP-Term in PSB1C3 :  
+
*** NarG with RBS-Amil CP-Term in pSB1C3 :  
-
** 45µL of Mix A+1µL of oligo 41 +2µL of DNA
+
**** 45µL of Mix A+1µL of oligo 41 +2µL of DNA
-
* NarK with RBS-LacZ-Term in PSB1C3 :  
+
*** NarK with RBS-LacZ-Term in pSB1C3 :  
-
** 45µL of Mix A+1µL of oligo 47+2µL of DNA
+
**** 45µL of Mix A+1µL of oligo 47+2µL of DNA
-
* NarK with RBS-Amil CP-Term in PSB1C3 :  
+
*** NarK with RBS-Amil CP-Term in pSB1C3 :  
-
** 45µL of Mix A+1µL of oligo 47 +2µL of DNA
+
**** 45µL of Mix A+1µL of oligo 47 +2µL of DNA
* Mix B :  
* Mix B :  
Line 42: Line 47:
** Oligo 43 : 25µL
** Oligo 43 : 25µL
-
* NirB with RBS-LacZ-Term in PSB1C3 :  
+
*** NirB with RBS-LacZ-Term in pSB1C3 :  
-
** 23µL of Mix B+2µL of DNA
+
**** 23µL of Mix B+2µL of DNA
-
* NirB with RBS-Amil CP-Term in PSB1C3 :  
+
*** NirB with RBS-Amil CP-Term in pSB1C3 :  
-
** 23µL of Mix B+2µL of DNA
+
**** 23µL of Mix B+2µL of DNA
-
* NarG with RBS-LacZ-Term in PSB1C3 :  
+
*** NarG with RBS-LacZ-Term in pSB1C3 :  
-
** 23µL of Mix B+2µL of DNA
+
**** 23µL of Mix B+2µL of DNA
-
* NarG with RBS-Amil CP-Term in PSB1C3 :  
+
*** NarG with RBS-Amil CP-Term in pSB1C3 :  
-
** 23µL of Mix B+2µL of DNA
+
**** 23µL of Mix B+2µL of DNA
-
* NarK with RBS-LacZ-Term in PSB1C3 :  
+
*** NarK with RBS-LacZ-Term in pSB1C3 :  
-
** 23µL of Mix B+2µL of DNA
+
**** 23µL of Mix B+2µL of DNA
-
* NarK with RBS-Amil CP-Term in PSB1C3 :  
+
*** NarK with RBS-Amil CP-Term in pSB1C3 :  
-
** 23µL of Mix B+2µL of DNA
+
**** 23µL of Mix B+2µL of DNA
-
=====2 - Gel electrophoresis of the colony PCR products=====
 
-
{|
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PCR Program for NirB with RBS-LacZ-Term in pSB1C3, NarG with RBS-LacZ-Term in pSB1C3, NarK with RBS-LacZ-Term in pSB1C3 :
-
| 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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[[File:PsPCRLacZ2108.jpg|400px]]  
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*6µL DNA Ladder
+
 
-
*10µL sample per well
+
PCR Program for NirB with RBS-Amil CP-Term in pSB1C3, NarG with RBS-Amil CP-Term in pSB1C3, NarK with RBS-Amil CP-Term in pSB1C3 :
-
*Gel : 0.8%
+
 
-
|}
+
[[File:PsPCRAmilCP2108.jpg|400px]]
-
Expected size : 3583bp
+
====2 - Culture of BBa_K1155000====
-
====3 - Culture ====
 
Nguyen
Nguyen
-
*BBa_K115500 strains (FNR repressor)in 5ml LB with Chlorenphenicol  
+
We made culture of BBa_K115500 strains in 5ml LB with Chlorenphenicol.
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*strain MG1665 Δ fnr:km clone1 in 5ml LB with kanamicine
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We incubate it at 37°C over night with shake 180 rpm.
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Incubate at 37°C over night with shake 180 rpm.
+
===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3'''====
-
===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''====
+
====1 - Electrophoresis of the digestion of BBa_J04450 by EcoRI/PstI====
-
 
+
-
====1 - Electrophoresis of the digestion of Bba_J04450 by EcoRI/PstI====
+
XiaoJing
XiaoJing
{|
{|
-
| style="width:350px;border:1px solid black;" | [[]]
+
| style="width:350px;border:1px solid black;" | [[File:Psgel2108.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
-
* Well 2 : 17µL of Bba_J04450 digested by EcoRI/PstI+3µL of H2O+4µL of 6X loading buffer  
+
* Well 2 : 17µL of BBa_J04450 digested by EcoRI/PstI+3µL of H2O+4µL of 6X loading buffer  
* Gel : 1%
* Gel : 1%
|}
|}
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Expected sizes :  
Expected sizes :  
* PSB3K3 : 2750 bp
* PSB3K3 : 2750 bp
-
* GFP : ... NON VISIBLE SUR LE GEL ???????
 
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We obtain a fragment at the right size. We can purify it.
+
We obtained a fragment with the right size. We can purify it.
|}
|}
 +
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
-
==='''A - Aerobic/Anaerobic regulation system / B - PCB se nsing system'''===
+
====Objective : obtaining FNR and BphR2 proteins====
-
===='''1 - Gibson assembloy.'''====
+
-
Xiaojing
+
-
* RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
+
====1 - Transformation of RBS-BphR2, FNR, RBS-FNR in DH5α====
-
* FNR_part1, FNR part1 and plasmid PSB1C3
+
-
* RBS_FNR part1, FNR_part2 and plasmid PSB1C3
+
-
 
+
-
 
+
-
 
+
-
Sample Volume:
+
-
{|
+
-
 
+
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| style="width:700px;border:1px solid black;vertical-align:top;" |
+
-
*Tube 1 : 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.
+
 +
XiaoJing
 +
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
 +
{| border="1" align="center"
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 +
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 +
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 +
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Latest revision as of 01:16, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 21

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Colony PCR of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α

Damir, Xiaojing

The transformation was good. We will do a Colony PCR.

Colonies repiquée dans 10µL d'eau. We do 8 PCR for each ligation.

Used quantities :

  • Mix A :
    • Buffer Dream Taq : 250µL
    • dNTP : 50µL
    • Oligo 44 : 50µL
    • Dream Taq : 20µL
    • H2O : 1.88mL
      • NirB with RBS-LacZ-Term in pSB1C3 :
        • 45µL of Mix A+1µL of oligo 45+2µL of DNA
      • NirB with RBS-Amil CP-Term in pSB1C3 :
        • 45µL of Mix A+1µL of oligo 45 +2µL of DNA
      • NarG with RBS-LacZ-Term in pSB1C3 :
        • 45µL of Mix A+1µL of oligo 41+2µL of DNA
      • NarG with RBS-Amil CP-Term in pSB1C3 :
        • 45µL of Mix A+1µL of oligo 41 +2µL of DNA
      • NarK with RBS-LacZ-Term in pSB1C3 :
        • 45µL of Mix A+1µL of oligo 47+2µL of DNA
      • NarK with RBS-Amil CP-Term in pSB1C3 :
        • 45µL of Mix A+1µL of oligo 47 +2µL of DNA
  • Mix B :
    • Mix A : 1.125mL
    • Oligo 43 : 25µL
      • NirB with RBS-LacZ-Term in pSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NirB with RBS-Amil CP-Term in pSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NarG with RBS-LacZ-Term in pSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NarG with RBS-Amil CP-Term in pSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NarK with RBS-LacZ-Term in pSB1C3 :
        • 23µL of Mix B+2µL of DNA
      • NarK with RBS-Amil CP-Term in pSB1C3 :
        • 23µL of Mix B+2µL of DNA


PCR Program for NirB with RBS-LacZ-Term in pSB1C3, NarG with RBS-LacZ-Term in pSB1C3, NarK with RBS-LacZ-Term in pSB1C3 :

PsPCRLacZ2108.jpg

PCR Program for NirB with RBS-Amil CP-Term in pSB1C3, NarG with RBS-Amil CP-Term in pSB1C3, NarK with RBS-Amil CP-Term in pSB1C3 :

PsPCRAmilCP2108.jpg

2 - Culture of BBa_K1155000

Nguyen

We made culture of BBa_K115500 strains in 5ml LB with Chlorenphenicol. We incubate it at 37°C over night with shake 180 rpm.

Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in pSB3K3

1 - Electrophoresis of the digestion of BBa_J04450 by EcoRI/PstI

XiaoJing

Psgel2108.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 17µL of BBa_J04450 digested by EcoRI/PstI+3µL of H2O+4µL of 6X loading buffer
  • Gel : 1%

Expected sizes :

  • PSB3K3 : 2750 bp

We obtained a fragment with the right size. We can purify it.

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

Objective : obtaining FNR and BphR2 proteins

1 - Transformation of RBS-BphR2, FNR, RBS-FNR in DH5α

XiaoJing

Protocol : Bacterial transformation


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