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Team:EPF-Lausanne/Notebook/Wet Lab
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Revision as of 14:31, 9 July 2009
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 | Characteristic | Resistance | Well (Kit Plate) |
|---|---|---|---|
|
[http://partsregistry.org/Part:BBa_B0010 BBa_B0010] |
Terminator |
A |
13D (1) |
|
[http://partsregistry.org/Part:BBa_R0010 BBa_R0010(promoter LacI)] |
A |
1D (1) | |
|
[http://partsregistry.org/Part:BBa_B0030 BBa_B0030(RBS)] |
A |
1H (1) | |
|
[http://partsregistry.org/Part:BBa_E0240 BBa_E0240(RBS-GFP-TER)] |
A |
12M (1) | |
|
[http://partsregistry.org/Part:BBa_I13507 BBa_I13507(RBS-mRFP-TER)] |
A |
22O (1) | |
|
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002(pTetR-RBS)] |
A |
13B (1) |
09.07.09
1. Miniprep and isolations of the yesterday 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. An agarose gel was runned to check PCR products
4. PCR products were digested with EcorI and SpeI and BBa_B0010(plasmid chosen containing the terminator) 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 (plasmid chosen containing the 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) |
Remarks: BBa_P1010, the death gene has to be transformed in ccdB (death gene) resistant cells: One Shot ccdB survival 2T1 E. Coli
