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 [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

Miniprep of the yesterday transformations were done, glycerol stock of [http://partsregistry.org/Part:BBa_I6007 Inverter TetR (BBa_I6007)] and [http://partsregistry.org/Part:BBa_P1010 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 [http://partsregistry.org/Part:BBa_I6007 Inverter TetR (BBa_I6007)] and [http://partsregistry.org/Part:BBa_P1010 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: [http://partsregistry.org/Part:BBa_P1010 Death Cassette (BBa_P1010)] sample contain the correct plasmid, [http://partsregistry.org/Part:BBa_I6007 Inverter TetR (BBa_I6007)] doesn't.

Finally, a gel extraction was done to purify the digested [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)] (linearized plasmid)

13.07.09

[http://partsregistry.org/Part:BBa_I6007 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 [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)], do it again tomorrow, beginning with the digestion, etc.

14.07.09

The digestion of the [http://partsregistry.org/Part:BBa_B0010 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 [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)] was runned on an agarose gel and purified with a gel extraction kit.

Even though linearized [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)] concentration was very low, we tried to ligate the previously amplified LOVTAP (08.07.09) in linearized [http://partsregistry.org/Part:BBa_B0010 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.

17.07.09

20.07.09

August