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 | |
