Paris/July 14

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'''Bastille's Day!!!'''
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Revision as of 14:48, 26 July 2007

Bastille's Day!!!

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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 Ampicilline medium

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

either LB or 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 (0.5 and 2mM).

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

PCRs

These two first PCRs aims at removing the Pst1 site in DGAT gene.

PCR : fw-DGAT1 rev-deltaPst1
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 27 forDGAT1 1µL YES
2m00' Oligo R 10µM 13 DPst1-DGAT-R 1µL Image (click to enlarge)
Number cycles Water 34µL Paris 14 07-DGAT.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA plasmid pKs::DGAT 1
PCR : fw_deltaPst1 rev-DGAT1
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 12 fw_deltaPst1 1µL YES
2m00' Oligo R 10µM 28 RevDGAT1 1µL Image (click to enlarge)
Number cycles Water 34µL Paris 14 07-DGAT.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA plasmid pKs::DGAT 2
PCR : Lox71-FtsA-FtsZ-1
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 3 Lox71-FtsA-F 2.5µL YES
2m00' Oligo R 10µM 4 DEcoR1-FtsZ-R 2.5µL Image (click to enlarge)
Number cycles Water 34µL Paris 15 07-Gel2.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA toothpick in glycerol stock of MG1655 1-2


PCR : FtsZ-2
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 5 DEcoR1-FtsZ-F 10µM 2.5µL YES
2m00' Oligo R 10µM 2 FtsZ-R 10µM 2.5µL Image (click to enlarge)
Number cycles Water 34µL Paris 15 07-Gel2.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA toothpick in glycerol stock of MG1655 3-4
PCR : Lox66-DapAColi
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 6 Lox66-DapAColi-F 10µM 2.5µL No
2m00' Oligo R 10µM 7 DapAColi-R 10µM 2.5µL Image (click to enlarge)
Number cycles Water 34µL Paris 15 07-Gel2.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA toothpick in glycerol stock of MG1655 5-6