Team:Paris Saclay/Notebook/August/21

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(→2 - Gel electrophoresis of the colony PCR products)
(→1 - Electrophoresis of the digestion of BBa_J04450 by EcoRI/PstI)
 
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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Obtaining FNR Promoter (nirB,narG,narK)plus  RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus  RBS_Amicip+Term_PSB1C3 '''====
+
===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
=====1 - Colony PCR on e.coli with FNR Promoter (nirB,narG,narK)plus  RBS_LacZ+Term_PSB1C3 and FNR Promoter (nirB,narG,narK)plus  RBS_Amicip+Term_PSB1C3  for 8 colonies of each=====
+
====1 - Colony PCR of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α====
-
Damir,Xiaojing
+
-
*A1-8: FNR Promoter nirB plus  RBS_LacZ+Term_PSB1C3
+
Damir, Xiaojing
-
*B1-8: FNR Promoter nirB plus  RBS_Amicip+Term_PSB1C3
+
-
*C1-8: FNR Promoter narG plus  RBS_LacZ+Term_PSB1C3
+
-
*D1-8: FNR Promoter narG plus  RBS_Amicip+Term_PSB1C3
+
-
*E1-8: FNR Promoter narK plus  RBS_LacZ+Term_PSB1C3
+
-
*F1-8: FNR Promoter narK Plus  RBS_Amicip+Term_PSB1C3
+
-
*Picking of 48 colonies
+
-
*Preparation  Master mix A
+
{|
-
**H<sub>2</sub>O : 1880µL
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
**dNTP : 50µL
+
The transformation was good. We will do a Colony PCR.
-
**VR primer : 50µL
+
|}
-
**DreamTaq buffer 10x : 250µL
+
-
**DreamTaq enzyme : 20µL
+
-
*Preparation  Master mix B
+
-
**Master mix A :1125µL
+
-
**VF2 primer : 25µL
+
 +
Colonies repiquée dans 10µL d'eau.
 +
We do 8 PCR for each ligation.
-
Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]] By using  Master mix B
+
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
-
=====2 - Gel electrophoresis of the colony PCR products=====
+
*** 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
-
{|
 
-
| style="width:350px;border:1px solid black;" | [[]]
 
-
| style="width:350px;border:1px solid black;vertical-align:top;" |
 
-
*6µL DNA Ladder
 
-
*10µL sample per well
 
-
*Gel : 0.8%
 
-
|}
 
-
Expected size : 3583bp
+
PCR Program for NirB with RBS-LacZ-Term in pSB1C3, NarG with RBS-LacZ-Term in pSB1C3, NarK with RBS-LacZ-Term in pSB1C3 :
-
====3 - Culture ====
+
[[File:PsPCRLacZ2108.jpg|400px]]
-
Nguyen
+
-
*BBa_K115500 strains (FNR repressor)in 5ml LB with Chlorenphenicol
+
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 :
-
*strain MG1665 Δ fnr:km clone1 in 5ml LB with kanamicine
+
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Incubate at 37°C over night with shake 180 rpm.
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[[File:PsPCRAmilCP2108.jpg|400px]]
-
====4 - Extract plasmid from strain BBa_K115500 clone1 by kit ====
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====2 - Culture of BBa_K1155000====
-
obtain plasmid 50µL concentration 195ng/µL
+
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.
-
=====5 - Digestion of Bba_K1155000 by SpeI =====
+
===='''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====
-
* Digestion by SpeI :
+
XiaoJing
-
**Buffer : 2µL
+
-
**SpeI : 2µL
+
-
**Plasmid : 10µL
+
-
**H20 : 6µL
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-
Do 3 tubes, Incubate at 37°C for 2 hours
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{|
 +
| style="width:350px;border:1px solid black;" | [[File:Psgel2108.jpg]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* 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
-
/
+
{|
-
+
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
==='''A - Aerobic/Anaerobic regulation system / B - PCB se nsing system'''===
+
We obtained a fragment with the right size. We can purify it.
-
===='''1 - Gibson assembloy.'''====
+
|}
-
Xiaojing
+
-
* RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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* FNR_part1, FNR part1 and plasmid PSB1C3
+
-
* RBS_FNR part1, FNR_part2 and plasmid PSB1C3
+
 +
====Objective : obtaining FNR and BphR2 proteins====
 +
====1 - Transformation of RBS-BphR2, FNR, RBS-FNR in DH5α====
-
Sample Volume:
+
XiaoJing
-
{|
+
-
 
+
-
| style="width:700px;border:1px solid black;vertical-align:top;" |
+
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*Tube 1 : BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
+
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*Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
+
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*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.
+
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Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.
+
 +
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/August/20|<big>Previous day</big>]]
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
|[[Team:Paris Saclay/Notebook/August/22|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
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Latest revision as of 01:16, 5 October 2013

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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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