Paris/July 14
From 2007.igem.org
yesterday -- tomorrow
Bastille's Day!!!
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 overnight 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 overnight in LB Ampicilline medium.
The culture (using a toothpick) was deposited on several solid LB media for growth:
+/- 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)
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 | ||||
