Team:Paris Saclay/Notebook/August/26

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For promoter fnr(activator)narG Digested SPE I :
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: Incubate at 37°C for 30 mins.
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:Then we performed a precipitation by ethanol in order to inactivate the enzymes for those 9 digestion.
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Protocol : [[Team:Paris_Saclay/Protocols/Ethanl precipitation|Ethanl precipitation]]
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
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===='''1 - Gibson assembly.'''====
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* RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
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* FNR_part1, FNR part1 and plasmid PSB1C3
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* RBS_FNR part1, FNR_part2 and plasmid PSB1C3
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Sample Volume:
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*Tube 1 : RBS_BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
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*Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
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*Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
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These 3 mixture is incubated at 50°C for up to one hour.
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Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.
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:Then we performed a precipitation by ethanol in order to inactivate the enzymes. And suspended the deposit by:
 
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|2µl ligation buffer
 
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|1µl ligase T4
 
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|1µl ligase T4+2µl PSB1C3
 
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|17µl H2O
 
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|15µl H2O
 
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:The incubation was during 1H30.
 

Revision as of 08:26, 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



h

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.





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


  • 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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