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Team:EPF-Lausanne/Notebook/Wet Lab
From 2009.igem.org
Contents |
Wet Lab
July
06.07.09
LOVTAP plasmid AND TrpR plasmid were transformed in competent E. Coli following received protocol, and grown overnight (see Lab book for more details).
One problem: we actually don't know TrpR plasmid resistance, so we tried with three resistances available in the lab: Amp., Kana. and Chl.
LOVTAP is in a plasmid called pCal-n (see picture below):
Some comments on the plasmid:
-CBP is a small peptide with which we could purify LOVTAP protein
-Thrombin target is a nucleotidic sequence that can be recognized by thrombin a peptidase. This peptidase will cut CBP once LOVTAP is purified
07.07.09
We have to grow the 3 strains generously sent by Tom Beatty
The three strains are :
- R.Palustris CEA001 (wild type) ; should be grown on LB medium only
- R.Palustris BPHP1+ ; should be grown on LB with gentamycin (100 micrograms/ml)
- E.Coli DH10B (pBPH/hmu0) ; should be grown on LB with gentamycin (20 micorgrams/ml)
The transformed LOVTAP and TrpR worked well (N.B. the plasmid of TrpR is pUC19 so the antibiotic resistance is Amp -> see below)
We did the glycerol stock, located in the -80 fridge, first floor of the iGEM compartement.
Then, a miniprep was done with both cultures. A LOVTAP plasmid aliquot was done, a TrpR plasmid aliquot was done, located in the -20 fridge, 2nd floor.
08.07.09
1. R. Palustris culture grew. A glycerol stock has been done. A pellet is on the fridge level 2, waiting for a miniprep.
2. iGEM parts have been transformed:
| Part | Resistance | Well (Kit Plate) |
|---|---|---|
|
BBa_B0010 (terminator) |
A |
13D (1) |
|
BBa_R0010 (promoter LacI) |
A |
1D (1) |
|
BBa_B0030 (RBS) |
A |
1H (1) |
|
BBa_E0240 (RBS-GFP-TER) |
A |
12M (1) |
|
BBa_I13507(RBS-mRFP-TER) |
A |
22O (1) |
|
BBa_J13002(pTetR-RBS) |
A |
13B (1) |
09.07.09
1. Miniprep and isolations of the yesterdey transformed plasmids. (cf. 08.09.09 subpart) Concentrations of the plasmids: cf. lab notebook pp. 8-9
2. PCR of pCal-n to PCR up LOVTAP with the different primers designed the 06.09.09, the products are:
- Prom_T7-RBS-CBP-LOVTAP
- RBS-CBP-LOVTAP
- CBP-LOVTAP
- LOVTAP
3. A gel was runned to check PCR products
4. PCR products were digested with EcorI and SpeI and BBa_B0010 was digested with EcorI and XbaI, the digestion products were treated with phosphatase. Then, PCR products were purified.
Finally, LOVTAP (PCR products) were ligated on BBa_B0010 (a plasmid containing a terminator).
5. Two more iGEM parts have been transformed:
| Part | Resistance | Well (Kit Plate) |
|---|---|---|
|
BBa_I6007 (inverter TetR) |
A |
1C (2) |
|
BBa_P1010 (death gene) |
C |
5E (1) |
