Team:Paris Saclay/Notebook/August/26

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
 +
='''Notebook : August 26'''=
='''Notebook : August 26'''=
-
 
-
=='''summary'''==
 
-
 
-
*Digestion for promoter fnr(repressor and activator) in PSB1C3 by ensyme SpeI and PstI.
 
-
 
-
*Ligation for promoter fnr(repressor or activator) in PSB1C3 digested by ensyme SPE I and PstI and RBS_LacZ+Term_or RBS_AmilCP+Term digested by PstI and XbeI.Transformation and incubated over night.
 
-
 
-
*3 Gibson assembly for  RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 and FNR_part1, FNR part1 and plasmid PSB1C3 and RBS_FNR part1, FNR_part2 and plasmid PSB1C3.Transformation and incubated over night.
 
-
<br>
 
-
 
-
=='''lab work'''==
 
-
 
-
 
-
 
-
:For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3  :
 
-
 
-
{| border="1" align="center"
 
-
|-
 
-
|promoter fnr(repressor)
 
-
|5µl
 
-
|-
 
-
|RBS_LacZ+Term
 
-
|3µl
 
-
|-
 
-
|Ligation buffer
 
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|2µl
 
-
|-
 
-
|Ligation T4 enzyme
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|8µl
 
-
|}
 
-
 
-
:For promoter fnr(repressor)+ RBS_AmilCP+Term_PSB1C3  :
 
-
 
-
{| border="1" align="center"
 
-
|-
 
-
|promoter fnr(repressor)
 
-
|5µl
 
-
|-
 
-
|RBS_AmilCP+Term
 
-
|3µl
 
-
|-
 
-
|Ligation buffer
 
-
|2µl
 
-
|-
 
-
|Ligation T4 enzyme
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|8µl
 
-
|}
 
-
 
-
:For promoter fnr(activator)nirB clone 6+ RBS_LacZ+Term_PSB1C3  :
 
-
 
-
{| border="1" align="center"
 
-
|-
 
-
|promoter fnr(activator)nirB clone 6
 
-
|5µl
 
-
|-
 
-
|RBS_LacZ+Term
 
-
|3µl
 
-
|-
 
-
|Ligation buffer
 
-
|2µl
 
-
|-
 
-
|Ligation T4 enzyme
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|8µl
 
-
|}
 
-
 
-
:For promoter fnr(activator)nirB clone 6+ RBS_AmilCP+Term_PSB1C3  :
 
-
 
-
{| border="1" align="center"
 
-
|-
 
-
|promoter fnr(activator)nirB clone 6
 
-
|5µl
 
-
|-
 
-
|RBS_AmilCP+Term
 
-
|3µl
 
-
|-
 
-
|Ligation buffer
 
-
|2µl
 
-
|-
 
-
|Ligation T4 enzyme
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|8µl
 
-
|}
 
-
<br>
 
-
 
-
Transformation these 4 ligation and spread in 8 LB plates with Chlorenphenicol . Incubate at 37°C over night.
 
-
 
-
 
-
 
-
 
-
*'''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''
 
-
**''1 - Gibson assembly.''
 
-
 
-
 
-
* RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
 
-
* 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;" |
 
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*Tube 1 : RBS_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.
 
-
 
-
 
-
 
-
 
-
 
-
 
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{| border="1" align="center"
 
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||<big>Previous week</big>
 
-
 
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|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 
-
 
-
|[[Team:Paris Saclay/Notebook/July/2|<big>Next day</big>]]
 
-
|}
 
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-
 
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{{Team:Paris_Saclay/incl_fin}}
 
-
 
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-
 
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{{Team:Paris_Saclay/incl_debut_generique}}
 
-
 
-
='''Notebook : August 20'''=
 
=='''Lab work'''==
=='''Lab work'''==
Line 153: Line 7:
==='''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 and 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
Line 165: Line 19:
** 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 171: 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 177: 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 184: 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 190: 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 197: 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 203: 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 209: 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 215: Line 69:
** H2O : 7µL
** H2O : 7µL
-
We let digestions 30 minutes at 37°C.
+
We incubate the digestion for 30 minutes at 37°C.
-
===='''2 - Denaturation of SpeI and 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
-
Protocol : [[Team:Paris_Saclay/Protocols/Ethanol precipitation|Ethanol precipitation]]
+
Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]]
We used 20µL of DNA.
We used 20µL of DNA.
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
-
* NarG clone 6 : 13.4ng/µl
+
* NarG clone 6 : 13.4ng/µL
-
* NarG clone 7 : 103.9ng/µl
+
* NarG clone 7 : 103.9ng/µL
-
* narG already digested by SpeI : 17.4ng/µl
+
* narG already digested by SpeI : 17.4ng/µL
-
* NirB clone 6 : 46ng/µl
+
* NirB clone 6 : 46ng/µL
-
* NirB clone 7 : 61.6ng/µl
+
* NirB clone 7 : 61.6ng/µL
-
* NirB already digested by SpeI : 16.1ng/µl
+
* NirB already digested by SpeI : 16.1ng/µL
{|
{|
| 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 decides 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
 +
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 : [[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'''===
 +
 +
===='''Objective : obtaining FNR and BphR2'''====
 +
 +
===='''1 - Gibson assembly'''====
 +
 +
XiaoJing
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
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 : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
 +
 +
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/August/23|<big>Previous day</big>]]
 +
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 +
|[[Team:Paris Saclay/Notebook/August/27|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 22:16, 4 October 2013

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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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