Team:Paris Saclay/Notebook/August/26

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(3 - Ligation of Pfnr, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3)
 
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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 - Digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI/PstI'''====
+
===='''1 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI/PstI'''====
XiaoJing  
XiaoJing  
-
* Bba_K1155000 :  
+
* BBa_K1155000 :  
** DNA : 14µL
** DNA : 14µL
** SpeI FD : 2µL
** SpeI FD : 2µL
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** Buffer FD : 2µL
** Buffer FD : 2µL
-
* Bba_K1155004 clone 6 :
+
* BBa_K1155004 clone 6 :
** DNA : 14µL
** DNA : 14µL
** SpeI FD : 2µL
** SpeI FD : 2µL
Line 25: Line 25:
** Buffer FD : 2µL
** Buffer FD : 2µL
-
* Bba_K1155004 clone 7 :
+
* BBa_K1155004 clone 7 :
** DNA : 14µL
** DNA : 14µL
** SpeI FD : 2µL
** SpeI FD : 2µL
Line 31: Line 31:
** Buffer FD : 2µL
** Buffer FD : 2µL
-
* Bba_K1155005 clone 6 :  
+
* BBa_K1155005 clone 6 :  
** DNA : 7µL
** DNA : 7µL
** SpeI FD : 2µL
** SpeI FD : 2µL
Line 38: Line 38:
** H2O : 7µL
** H2O : 7µL
-
* Bba_K1155005 clone 7 :
+
* BBa_K1155005 clone 7 :
** DNA : 14µL
** DNA : 14µL
** SpeI FD : 2µL
** SpeI FD : 2µL
Line 44: Line 44:
** Buffer FD : 2µL
** Buffer FD : 2µL
-
* Bba_K1155006 clone 6 :
+
* BBa_K1155006 clone 6 :
** DNA : 4µL
** DNA : 4µL
** SpeI FD : 2µL
** SpeI FD : 2µL
Line 51: Line 51:
** H2O : 10µL
** H2O : 10µL
-
Bba_K1155004 already digested by SpeI :  
+
* BBa_K1155004 already digested by SpeI :  
** DNA : 13µL
** DNA : 13µL
** PstI FD : 2µL
** PstI FD : 2µL
Line 57: Line 57:
** H2O : 3µL
** H2O : 3µL
-
Bba_K1155005 already digested by SpeI :  
+
* BBa_K1155005 already digested by SpeI :  
** DNA : 10µL
** DNA : 10µL
** PstI FD : 2µL
** PstI FD : 2µL
Line 63: Line 63:
** H2O : 6µL
** H2O : 6µL
-
* Bba_K1155006 already digested by SpeI :  
+
* BBa_K1155006 already digested by SpeI :  
** DNA : 9µL
** DNA : 9µL
** PstI FD : 2µL
** PstI FD : 2µL
Line 69: Line 69:
** H2O : 7µL
** H2O : 7µL
-
We keep digestions for 30 minutes at 37°C.
+
We incubate the digestion for 30 minutes at 37°C.
-
===='''2 - Denaturation of SpeI/PstI used for the digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+
===='''2 - Inactivation of SpeI/PstI used for the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
XiaoJing
XiaoJing
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Nanodrop :  
Nanodrop :  
-
* Pfnr : 91.8ng/µL
+
* Pndh* : 91.8ng/µL
* NarK clone 6 : 19.8ng/µL
* NarK clone 6 : 19.8ng/µL
* Nark already digested by SpeI : 15.1ng/µL
* Nark already digested by SpeI : 15.1ng/µL
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
According to the result of the Nanodrop, we decide to do ligations with Pfnr and nirB clone 6.
+
According to the result of the Nanodrop, we decide to do ligations with Pndh* and nirB clone 6.
|}
|}
-
===='''3 - Ligation of Pfnr, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3'''====
+
===='''3 - Ligation of Pndh*, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3'''====
XiaoJing
XiaoJing
Line 101: Line 101:
Used quantities :  
Used quantities :  
-
NirB clone 6 with RBS-LacZ or RBS-Amil CP, Pfnr with RBS-LacZ or RBS-Amil CP :
+
NirB clone 6 with RBS-LacZ or RBS-Amil CP, Pndh* with RBS-LacZ or RBS-Amil CP :
* RBS-LacZ-Term or RBS-Amil CP-Term : 3µL
* RBS-LacZ-Term or RBS-Amil CP-Term : 3µL
-
* Pfnr or NirB clone 6 : 5µL  
+
* Pndh* or NirB clone 6 : 5µL  
* Buffer ligase : 2µL
* Buffer ligase : 2µL
* Ligase T4 : 2µL
* Ligase T4 : 2µL
* H20 : 8µL
* H20 : 8µL
-
We keep the ligation for 1h at 37°C.
+
We incubate the ligation for 1h at 37°C.
===='''4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3'''====
===='''4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3'''====
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Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
 +
 +
===='''5- Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20'''====
 +
 +
XioaJing
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
Transformation of 08/19/13 works. We will purify our colonies.
 +
|}
 +
 +
We streaked colonies into ampicilin and chloramphenicol plates and incubate at 30°C.
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===

Latest revision as of 22:16, 4 October 2013

Contents

Notebook : August 26

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI/PstI

XiaoJing

  • BBa_K1155000 :
    • DNA : 14µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
  • BBa_K1155004 clone 6 :
    • DNA : 14µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
  • BBa_K1155004 clone 7 :
    • DNA : 14µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
  • BBa_K1155005 clone 6 :
    • DNA : 7µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 7µL
  • BBa_K1155005 clone 7 :
    • DNA : 14µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
  • BBa_K1155006 clone 6 :
    • DNA : 4µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 10µL
  • BBa_K1155004 already digested by SpeI :
    • DNA : 13µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 3µL
  • BBa_K1155005 already digested by SpeI :
    • DNA : 10µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 6µL
  • BBa_K1155006 already digested by SpeI :
    • DNA : 9µL
    • PstI FD : 2µL
    • Buffer FD : 2µL
    • H2O : 7µL

We incubate the digestion for 30 minutes at 37°C.

2 - Inactivation of SpeI/PstI used for the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

XiaoJing

Protocol : Ethanol precipitation

We used 20µL of DNA.

Nanodrop :

  • Pndh* : 91.8ng/µL
  • NarK clone 6 : 19.8ng/µL
  • Nark already digested by SpeI : 15.1ng/µL
  • NarG clone 6 : 13.4ng/µL
  • NarG clone 7 : 103.9ng/µL
  • narG already digested by SpeI : 17.4ng/µL
  • NirB clone 6 : 46ng/µL
  • NirB clone 7 : 61.6ng/µL
  • NirB already digested by SpeI : 16.1ng/µL

According to the result of the Nanodrop, we decide to do ligations with Pndh* and nirB clone 6.

3 - Ligation of Pndh*, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3

XiaoJing

Used quantities :

NirB clone 6 with RBS-LacZ or RBS-Amil CP, Pndh* with RBS-LacZ or RBS-Amil CP :

  • RBS-LacZ-Term or RBS-Amil CP-Term : 3µL
  • Pndh* or NirB clone 6 : 5µL
  • Buffer ligase : 2µL
  • Ligase T4 : 2µL
  • H20 : 8µL

We incubate the ligation for 1h at 37°C.

4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3

XiaoJing

Protocol : Bacterial transformation

5- Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20

XioaJing

Transformation of 08/19/13 works. We will purify our colonies.

We streaked colonies into ampicilin and chloramphenicol plates and incubate at 30°C.

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

Objective : obtaining FNR and BphR2

1 - Gibson assembly

XiaoJing

Tranformation of 08/21 didn't work. We will do the Gibson assembly again.

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 keep these mix at 50°C for 1h.

2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α

XiaoJing

Protocol : Bacterial transformation


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