Team:Paris Saclay/Notebook/August/26

From 2013.igem.org

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:According to the result of nanodrop we decide to do 4 ligation :<br>
:According to the result of nanodrop we decide to do 4 ligation :<br>
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 +
:For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3  :
{| border="1" align="center"
{| border="1" align="center"
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|promoter fnr(repressor)
|promoter fnr(repressor)
|5µl
|5µl
 +
|-
|RBS_LacZ+Term
|RBS_LacZ+Term
|3µl
|3µl
|-
|-
|Ligation buffer
|Ligation buffer
 +
|2µl
 +
|-
 +
|Ligation T4 enzyme
|2µl
|2µl
|-
|-
|H2O
|H2O
-
|14µl
+
|8µl
-
|}<br>
+
|}
 +
: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>

Revision as of 08:58, 9 September 2013

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Contents

Notebook : August 26

summary

  • started the Restriction digestion and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoRI and PstI.
  • Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which was created by iGEM UCL team in 2009
  • Designed oligonucleotides for the gene coding for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group


lab work


  • A.aero/anaerobic regulation system
    • 1.BioBrick promotor fnr(repressor and activator) in plasmid PSB1C3


Digestion for promoter fnr(repressor and activator) in PSB1C3


2 enzymes SPE I and PST I
For promoter fnr(repressor) :
DNA 14µl
SPE I 2µl
PST I 2µl
H2O 0µl
buffer 2µl
total 20µl


For promoter fnr(activator)nirB clone 6:
DNA 14µl
SPE I 2µl
PST I 2µl
H2O 0µl
buffer 2µl
total 20µl
For promoter fnr(activator)nark clone 6 :
DNA 4µl
SPE I 2µl
PST I 2µl
H2O 10µl
buffer 2µl
total 20µl
For promoter fnr(activator)nirB clone 7 :
DNA 14µl
SPE I 2µl
PST I 2µl
H2O 0µl
buffer 2µl
total 20µl
For promoter fnr(activator)narG clone 6 :
DNA 7µl
SPE I 2µl
PST I 2µl
H2O 7µl
buffer 2µl
total 20µl
For promoter fnr(activator)narG clone 7 :
DNA 14µl
SPE I 2µl
PST I 2µl
H2O 0µl
buffer 2µl
total 20µl


For promoter fnr(activator)nark Digested SPE I :
DNA 9µl
SPE I 0µl
PST I 2µl
H2O 7µl
buffer 2µl
total 20µl
For promoter fnr(activator)narB Digested SPE I :
DNA 13µl
SPE I 0µl
PST I 2µl
H2O 3µl
buffer 2µl
total 20µl



For promoter fnr(activator)narG Digested SPE I :
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 : Ethanl precipitation


Degestion product quantification
We used the nano drop to measure the DNA in 260nm and we found its concentration
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


Ligation
According to the result of nanodrop we decide to do 4 ligation :
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




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.







  • B.PCBs sensor system
    • 1.BioBrick BphR2(regulator) in plasmid PSB1C3


Using software gene manager to find the oligopeptide for amplification of BphR2.


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