From 2007.igem.org

Bastille's Day!!!

Contents 1 What to do ? 2 What has been done 3 DGAT expressing E.coli 4 PCRs

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
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
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
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
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
30		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	toothpick in glycerol stock of MG1655			5-6