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Revision as of 16:13, 15 July 2007 (view source) Eismoustique (Talk | contribs) (→What has been done) ← Older edit		Revision as of 20:36, 15 July 2007 (view source) Eismoustique (Talk | contribs) (→What has been done) Newer edit →
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	** Minipreps to isolate the plasmids carrying the biobricks		** Minipreps to isolate the plasmids carrying the biobricks
	** Glycerol stocks of the transformed strains		** 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 ==		== PCRs ==

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Revision as of 20:36, 15 July 2007

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 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	toughpick 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	toughpick 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	toughpick in glycerol stock of MG1655