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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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
 +
='''Notebook : August 26'''=
='''Notebook : August 26'''=
-
=='''summary'''==
+
=='''Lab work'''==
-
*started the Restriction digestion and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoRI and PstI.
+
==='''A - Aerobic/Anaerobic regulation system'''===
-
*Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which was created by iGEM UCL team in 2009
+
===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
*Designed oligonucleotides for the gene coding for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group
+
===='''1 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI/PstI'''====
-
<br>
+
-
=='''lab work'''==
+
XiaoJing
-
<br>
+
* BBa_K1155000 :
-
*'''A.aero/anaerobic regulation system'''<br>
+
** DNA : 14µL
-
**''1.BioBrick promotor fnr(repressor and activator) in plasmid PSB1C3''
+
** SpeI FD : 2µL
-
<br>
+
** PstI FD : 2µL
-
:<u>Digestion for promoter fnr(repressor and activator) in PSB1C3</u>
+
** Buffer FD : 2µL
-
<br>
+
-
:2 enzymes SPE I and PST I
+
-
:For promoter fnr(repressor) :
+
* BBa_K1155004 clone 6 :
 +
** DNA : 14µL
 +
** SpeI FD : 2µL
 +
** PstI FD : 2µL
 +
** Buffer FD : 2µL
-
{| border="1" align="center"
+
* BBa_K1155004 clone 7 :
-
|-o
+
** DNA : 14µL
-
|DNA
+
** SpeI FD : 2µL
-
|14µl
+
** PstI FD : 2µL
-
|-
+
** Buffer FD : 2µL
-
| SPE I
+
-
|2µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|H2O
+
-
|0µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
-
|}
+
 +
* BBa_K1155005 clone 6 :
 +
** DNA : 7µL
 +
** SpeI FD : 2µL
 +
** PstI FD : 2µL
 +
** Buffer FD : 2µL
 +
** H2O : 7µL
-
:For promoter fnr(activator)nirB clone 6:
+
* BBa_K1155005 clone 7 :
 +
** DNA : 14µL
 +
** SpeI FD : 2µL
 +
** PstI FD : 2µL
 +
** Buffer FD : 2µL
-
{|align="center" border="1"
+
* BBa_K1155006 clone 6 :
-
|-
+
** DNA : 4µL
-
|DNA
+
** SpeI FD : 2µL
-
|14µl
+
** PstI FD : 2µL
-
|-
+
** Buffer FD : 2µL
-
|SPE I
+
** H2O : 10µL
-
|2µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|H2O
+
-
|0µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
-
|}
+
-
:For promoter fnr(activator)nark clone 6 :
+
* BBa_K1155004 already digested by SpeI :  
 +
** DNA : 13µL
 +
** PstI FD : 2µL
 +
** Buffer FD : 2µL
 +
** H2O : 3µL
-
{| border="1" align="center"
+
* BBa_K1155005 already digested by SpeI :
-
|-o
+
** DNA : 10µL
-
|DNA
+
** PstI FD : 2µL
-
|4µl
+
** Buffer FD : 2µL
-
|-
+
** H2O : 6µL
-
| SPE I
+
-
|2µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|H2O
+
-
|10µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
-
|}
+
-
:For promoter fnr(activator)nirB clone 7 :
+
* BBa_K1155006 already digested by SpeI :  
 +
** DNA : 9µL
 +
** PstI FD : 2µL
 +
** Buffer FD : 2µL
 +
** H2O : 7µL
-
{| border="1" align="center"
+
We incubate the digestion for 30 minutes at 37°C.
-
|-o
+
-
|DNA
+
-
|14µl
+
-
|-
+
-
| SPE I
+
-
|2µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|H2O
+
-
|0µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
-
|}
+
-
+
-
:For promoter fnr(activator)narG clone 6 :
+
-
{| border="1" align="center"
+
===='''2 - Inactivation of SpeI/PstI used for the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
|-o
+
-
|DNA
+
-
|7µl
+
-
|-
+
-
| SPE I
+
-
|2µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|H2O
+
-
|7µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
-
|}
+
-
:For promoter fnr(activator)narG clone 7 :
+
XiaoJing
-
{| border="1" align="center"
+
Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]]
-
|-o
+
-
|DNA
+
-
|14µl
+
-
|-
+
-
| SPE I
+
-
|2µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|H2O
+
-
|0µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
-
|}
+
 +
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
-
:For promoter fnr(activator)nark Digested SPE I :
+
{|
-
 
+
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
{| border="1" align="center"
+
According to the result of the Nanodrop, we decide to do ligations with Pndh* and nirB clone 6.
-
|-oon
+
-
|DNA
+
-
|9µl
+
-
|-
+
-
| SPE I
+
-
|0µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|H2O
+
-
|7µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
|}
|}
-
:For promoter fnr(activator)narB Digested SPE I :
+
===='''3 - Ligation of Pndh*, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3'''====
-
{| border="1" align="center"
+
XiaoJing
-
|-oon
+
-
|DNA
+
-
|13µl
+
-
|-
+
-
| SPE I
+
-
|0µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|H2O
+
-
|3µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
-
|}
+
 +
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.
-
:For promoter fnr(activator)narG Digested SPE I :
+
===='''4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3'''====
-
{| border="1" align="center"
+
XiaoJing
-
|-oon
+
-
|DNA
+
-
|10µl
+
-
|-
+
-
| SPE I
+
-
|0µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|H2O
+
-
|6µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
-
|}
+
-
: Incubate at 37°C for 30 mins.
+
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
-
:Then we performed a precipitation by ethanol in order to inactivate the enzymes for those 9 digestion.
+
-
Protocol : [[Team:Paris_Saclay/Protocols/Ethanl precipitation|Ethanl precipitation]]
+
 +
===='''5- Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20'''====
 +
XioaJing
-
:<u>Degestion product quantification</u>
+
{|
-
 
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
:We used the nano drop to measure the DNA in 260nm and we found its concentration
+
Transformation of 08/19/13 works. We will purify our colonies.
-
{| border="1" align="center"
+
-
|-
+
-
|name
+
-
| fnr(repressor)
+
-
| fnr nirB clone 6
+
-
| fnr nark clone 6
+
-
| fnr nirB clone 7
+
-
| fnr narG clone 6
+
-
| fnr narG clone 7
+
-
| fnr nark Dig. SPE I
+
-
| fnr narB Dig. SPE I
+
-
| fnr narG Dig. SPE I
+
-
|-
+
-
|conc.
+
-
|91.8ng/µl
+
-
|46.0ng/µl
+
-
|19.8ng/µl
+
-
|61.6ng/µl
+
-
|13.4ng/µl
+
-
|103.9ng/µl
+
-
|15.1ng/µl
+
-
|16.1ng/µl
+
-
|17.1ng/µl
+
|}
|}
-
<br>
+
We streaked colonies into ampicilin and chloramphenicol plates and incubate at 30°C.
-
:<u>Ligation</u>
+
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
-
:According to the result of nanodrop we decide to do 4 ligation :<br>
+
===='''Objective : obtaining FNR and BphR2'''====
-
:For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3  :
+
===='''1 - Gibson assembly'''====
-
{| border="1" align="center"
+
XiaoJing
-
|-
+
-
|promoter fnr(repressor)
+
-
|5µl
+
-
|-
+
-
|RBS_LacZ+Term
+
-
|3µl
+
-
|-
+
-
|Ligation buffer
+
-
|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>
 
-
 
-
 
-
 
-
 
-
==='''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:
 
{|
{|
-
 
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
| style="width:700px;border:1px solid black;vertical-align:top;" |
+
Tranformation of 08/21 didn't work. We will do the Gibson assembly again.
-
*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.
 
 +
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
-
<br>
+
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]]
-
 
-
*'''B.PCBs sensor system'''<br>
 
-
**''1.BioBrick BphR2(regulator) in plasmid PSB1C3''<br>
 
-
 
-
 
-
:Using software gene manager to find the oligopeptide for amplification of BphR2.
 
-
 
-
<br>
 
{| border="1" align="center"
{| border="1" align="center"
-
||<big>Previous week</big>
+
|[[Team:Paris Saclay/Notebook/August/23|<big>Previous day</big>]]
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
-
|[[Team:Paris Saclay/Notebook/July/2|<big>Next day</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

Navigation Home Team Project Labwork Notebook Human practices

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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