Team:Paris Saclay/Notebook/July/1

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:Using software gene manager to find the oligopeptide for amplification of BphR2.
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Revision as of 03:20, 25 August 2013

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Notebook : July 1

summary

  • Commenced the Restriction digest and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoR I and PST I.
  • Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which is created by iGEM UCL team in 2009
  • Designed oligopeptides for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group


detail


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


Digestion for fnr and PSB1C3


2 enzymes EcoR I and PST I can be used in one common buffer: orange buffer (10X).
For PCR products:
PCR products 20µl
EcoR I 0.75µl
PST I 0.75µl
H2O 5.5µl
buffer 3µl
total 30µl


For plasmid PSB1C3:
Plasmid 4µl
EcoR I 0.5µl
PST I 0.5µl
H2O 2.2µl
buffer 0.8µl
total 8µl


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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