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):

pCal-n plasmid

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)

pUC19 plasmid

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:

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 [http://partsregistry.org/Part:BBa_B0010 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 [http://partsregistry.org/Part:BBa_B0010 BBa_B0010] (plasmid chosen containing the terminator).

5. Two more iGEM parts have been transformed:

Remarks: [http://partsregistry.org/Part:BBa_P1010 BBa_P1010], the death gene has to be transformed in ccdB (death gene) resistant cells: One Shot ccdB survival 2T1 E. Coli

10.07.09

August