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

Result: Prom_T7-RBS-CBP-LOVTAP didn't worked, the other were ok.

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:

Remarks: 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

Miniprep of the yesterday transformations were done, glycerol stock of Inverter TetR (BBa_I6007) and Death Cassette (BBa_P1010) were done and finally they were put at -80°C.

As Prom_T7-RBS-CBP-LOVTAP PCR product migration on the gel didn't worked (in fact there wasn't enough products) July the 9th, a second PCR was done with more cycles (40) to check wether the the primers were accurately designed.

Results: The primers are correct -> Prom_T7-RBS-CBP-LOVTAP was correctly amplified.

A digestion-ligation according iGEM special protocol Biobrick_Assembly_Manual was done with LacI and RBS. And Term was digested (with E and X) to linearize the plasmid.

As Inverter TetR (BBa_I6007) and Death Gene (BBa_P1010) were transformed with a contaminated SOC, we did a PCR with the isolated plasmids to check if the plasmids were correct.

Results: Death Cassette (BBa_P1010) sample contain the correct plasmid, Inverter TetR (BBa_I6007) doesn't.

Finally, a gel extraction was done to purify the digested Terminator (BBa_B0010) (linearized plasmid)

13.07.09

Inverter TetR (BBa_I6007) was transformed again. And the new plasmid (created on July the 10th, 10.07.09) LacI-RBS was transformed on DH5-alpha competent cells.

We failed in purifing our Terminator (BBa_B0010), do it again tomorrow, beginning with the digestion, etc.

14.07.09

The digestion of the Terminator (BBa_B0010) was done, in order to purify it once more, using gel extraction.

Miniprep of LacI-RBS and Inverter TetR, and PCR of the two of them.

The linearized Terminator (BBa_B0010) was runned on an agarose gel and purified with a gel extraction kit.

Even though linearized Terminator (BBa_B0010) concentration was very low, we tried to ligate the previously amplified LOVTAP (08.07.09) in linearized Terminator (BBa_B0010)

15.07.09

Miniprep of LacI-RBS (again, because we had a very low concentration yesterday). We obtained a better concentration.

Transformation of LovTAP-Term (LB + plates).

LB-Agar plates without antibiotic and with chlorophenicol & Ampicilin have been made.

16.07.09

Our plates and tubes from yesterday transformation are the same for the negative control and the LovTAP-Term. So either the ligation or the transformation didn't work. Actually we think that it is the transformation, because we put the wrong antibiotics : Chl instead of Amp. As we don't have any ligation products any more, we did the digestion, gel extraction and ligation again.

We also did a PCR of LacI-RBS product of the second transformation (of 15.07.09), as the concentration for the transformation of 14.07.09 was too low. We then ran the gel.

Gel extraction of Term.

Preparation of LB plates : AMP plates and Chl plates.

Then ligation of LovTAP and Term.

Finally digestion of the ligation product.

17.07.09

20.07.09

August