Team:Paris Saclay/Notebook/August/26

From 2013.igem.org

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Revision as of 08:13, 9 September 2013

Navigation Home Team Project Labwork Notebook Human practices

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)nark Digested SPE I :
DNA 9µl
SPE I 0µl
PST I 2µl
H2O 7µl
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)narG Digested SPE I :
DNA 10µl
SPE I 0µl
PST I 2µl
H2O 6µl
buffer 2µl
total 20µl





Ligation
After 3h of digestion, we mixed the digestion products:
PCR product 30µl
PSB1C3 4µl
Ligation buffer 2µl
H2O 14µl

Then we performed a precipitation by ethanol in order to inactivate the enzymes. And suspended the deposit by:
mixture control
2µl ligation buffer 2µl ligation buffer
1µl ligase T4 1µl ligase T4+2µl PSB1C3
17µl H2O 15µl H2O


The incubation was during 1H30.


  • 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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