Paris/July 14

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Revision as of 09:13, 16 July 2007 by David.bikard (Talk | contribs)

Contents

What to do ?

  • Remove the plate of the transduction experiment and the transformation ones from the 37°C incubator (Upper-left shelf): If it has worked and if someones comes on Sunday:
    • isolation of the clones from the transduction on petri-dishes (LB+DAP+Erm+Citrate)
    • LB + antibio culture of the transformants (to make a glycerol stock and to do MiniPreps)
  • Make a gel (0.8%) and migrate the PCR products
  • Purify them
  • Do the assembly PCR

If enough time (we can also wait to have the plasmids to do everything at the same time):

  • Digestion of the purifications products with appropriate enzymes
  • Purification


What has been done

  • Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121):
    • Isolation of 10 different colonies on LB Citrate Erythro DAP plates


  • Seeding LB-Amp cultures of the following strains
    • DH5alpha transformed with the plasmid carrying the Biobrick pJ23100
    • DH5alpha transformed with the plasmid carrying the Biobrick B0015

these two ON cultures will allow us to perform (tomorrow):

    • Minipreps to isolate the plasmids carrying the biobricks
    • Glycerol stocks of the transformed strains


DGAT expressing E.coli

  • After transformation of DH5alpha E.coli with pKS::DGAT plasmid, a transformant clone was grown ON in LB Erythromycin medium

The culture (using a toothpick)was deposited on several solid media for growth:

either LB or M9 minimal medium. +/- IPTG (inducer of DGAT expression, dgat gene being carried by pKS::DGAT plasmid) +/- Nile Red (fat detection dye) +/- oleate at 2 different concentrations.

This experiment aims at finding out if E.coli cells that express DGAT from Acinetobacter can incorporate oleate (a fatty acid, catalyse the reaction between a fatty acid and diacyl glycerol and finally accumulate fat in the form of triglyceride.

PCRs

PCR : Lox71-FtsA-FtsZ-1
Name Lox71-FtsA-FtsZ-1
Annealing T° 55°C
Time of elongation 2m00'
Number of Cycles 30
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 3 Lox71-FtsA-F 10µM 2.5µL
oligoR 4 DEcoR1-FtsZ-R 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA toothpick in glycerol stock of MG1655


PCR : FtsZ-2
Name FtsZ-2
Annealing T° 55°C
Time of elongation 2m00'
Number of Cycles 30
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 5 DEcoR1-FtsZ-F 10µM 2.5µL
oligoR 2 FtsZ-R 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA toothpick in glycerol stock of MG1655


PCR : Lox66-DapAColi
Name Lox66-DapAColi
Annealing T° 55°C
Time of elongation 2m00'
Number of Cycles 30
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 6 Lox66-DapAColi-F 10µM 2.5µL
oligoR 7 DapAColi-R 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA toothpick in glycerol stock of MG1655