Team:Paris Saclay/Notebook/August/26

From 2013.igem.org

(Difference between revisions)
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=='''lab work'''==
=='''lab work'''==
-
<br>
 
-
*'''A.aero/anaerobic regulation system'''<br>
 
-
**''1.BioBrick promotor fnr(repressor and activator) in plasmid PSB1C3''
 
-
<br>
 
-
:<u>Digestion for promoter fnr(repressor and activator) in PSB1C3</u>
 
-
<br>
 
-
:2 enzymes SPE I and PST I
 
-
 
-
:For promoter fnr(repressor) :
 
-
 
-
{| border="1" align="center"
 
-
|-o
 
-
|DNA
 
-
|14µl
 
-
|-
 
-
| SPE I
 
-
|2µl
 
-
|-
 
-
|PST I
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|0µl
 
-
|-
 
-
|buffer
 
-
|2µl
 
-
|-
 
-
|total
 
-
|20µl
 
-
|}
 
-
 
-
 
-
:For promoter fnr(activator)nirB clone 6:
 
-
 
-
{|align="center" border="1"
 
-
|-
 
-
|DNA
 
-
|14µl
 
-
|-
 
-
|SPE I
 
-
|2µl
 
-
|-
 
-
|PST I
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|0µl
 
-
|-
 
-
|buffer
 
-
|2µl
 
-
|-
 
-
|total
 
-
|20µl
 
-
|}
 
-
 
-
:For promoter fnr(activator)nark clone 6 :
 
-
 
-
{| border="1" align="center"
 
-
|-o
 
-
|DNA
 
-
|4µl
 
-
|-
 
-
| SPE I
 
-
|2µl
 
-
|-
 
-
|PST I
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|10µl
 
-
|-
 
-
|buffer
 
-
|2µl
 
-
|-
 
-
|total
 
-
|20µl
 
-
|}
 
-
 
-
:For promoter fnr(activator)nirB clone 7 :
 
-
 
-
{| border="1" align="center"
 
-
|-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"
 
-
|-o
 
-
|DNA
 
-
|7µl
 
-
|-
 
-
| SPE I
 
-
|2µl
 
-
|-
 
-
|PST I
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|7µl
 
-
|-
 
-
|buffer
 
-
|2µl
 
-
|-
 
-
|total
 
-
|20µl
 
-
|}
 
-
 
-
:For promoter fnr(activator)narG clone 7 :
 
-
 
-
{| border="1" align="center"
 
-
|-o
 
-
|DNA
 
-
|14µl
 
-
|-
 
-
| SPE I
 
-
|2µl
 
-
|-
 
-
|PST I
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|0µl
 
-
|-
 
-
|buffer
 
-
|2µl
 
-
|-
 
-
|total
 
-
|20µl
 
-
|}
 
-
 
-
 
-
 
-
:For promoter fnr(activator)nark Digested SPE I :
 
-
 
-
{| border="1" align="center"
 
-
|-oon
 
-
|DNA
 
-
|9µl
 
-
|-
 
-
| SPE I
 
-
|0µl
 
-
|-
 
-
|PST I
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|7µl
 
-
|-
 
-
|buffer
 
-
|2µl
 
-
|-
 
-
|total
 
-
|20µl
 
-
|}
 
-
 
-
:For promoter fnr(activator)narB Digested SPE I :
 
-
 
-
{| border="1" align="center"
 
-
|-oon
 
-
|DNA
 
-
|13µl
 
-
|-
 
-
| SPE I
 
-
|0µl
 
-
|-
 
-
|PST I
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|3µl
 
-
|-
 
-
|buffer
 
-
|2µl
 
-
|-
 
-
|total
 
-
|20µl
 
-
|}
 
-
 
-
 
-
 
-
 
-
:For promoter fnr(activator)narG Digested SPE I :
 
-
 
-
{| border="1" align="center"
 
-
|-oon
 
-
|DNA
 
-
|10µl
 
-
|-
 
-
| SPE I
 
-
|0µl
 
-
|-
 
-
|PST I
 
-
|2µl
 
-
|-
 
-
|H2O
 
-
|6µl
 
-
|-
 
-
|buffer
 
-
|2µl
 
-
|-
 
-
|total
 
-
|20µl
 
-
|}
 
-
 
-
: Incubate at 37°C for 30 mins.
 
-
: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]]
 
-
 
-
 
-
 
-
:<u>Degestion product quantification</u>
 
-
 
-
:We used the nano drop to measure the DNA in 260nm and we found its concentration
 
-
{| 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>
 
-
 
-
:<u>Ligation</u>
 
-
:According to the result of nanodrop we decide to do 4 ligation :<br>
 
:For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3  :
:For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3  :
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|[[Team:Paris Saclay/Notebook/July/2|<big>Next day</big>]]
|[[Team:Paris Saclay/Notebook/July/2|<big>Next day</big>]]
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{{Team:Paris_Saclay/incl_fin}}
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{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : August 20'''=
 +
 +
=='''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 and 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 let digestions 30 minutes at 37°C.
 +
 +
===='''2 - Denaturation of SpeI and PstI used for the digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
 +
 +
XiaoJing
 +
 +
Protocol : [[Team:Paris_Saclay/Protocols/Ethanol precipitation|Ethanol precipitation]]
 +
 +
We used 20µL of DNA.
 +
 +
Nanodrop :
 +
* Pfnr : 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
 +
 +
{|
 +
| 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.
 +
|}
 +
 +
===='''3 - Ligation of Pfnr, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3'''====
 +
 +
XiaoJing
 +
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 21:00, 20 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

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.


lab work

For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3  :
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  :
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  :
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  :
promoter fnr(activator)nirB clone 6 5µl
RBS_AmilCP+Term 3µl
Ligation buffer 2µl
Ligation T4 enzyme 2µl
H2O 8µl


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:

  • 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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Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 20

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 and 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 let digestions 30 minutes at 37°C.

2 - Denaturation of SpeI and PstI used for the digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006

XiaoJing

Protocol : Ethanol precipitation

We used 20µL of DNA.

Nanodrop :

  • Pfnr : 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 decides to do ligations with Pfnr and nirB clone 6.

3 - Ligation of Pfnr, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3

XiaoJing


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