Team:EPF-Lausanne/Notebook/Wet Lab

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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):

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:

Parts&Characteristics
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

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:

Parts&Characteristics
Part Characteristic Resistance Well (Kit Plate)

[http://partsregistry.org/Part:BBa_I6007 BBa_I6007]

Double repressor: called Inverter TetR

A

1C (2)

[http://partsregistry.org/Part:BBa_P1010 BBa_P1010]

Death Cassette

C

5E (1)


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.


16.07.09

17.07.09

20.07.09

August