Team:EPF-Lausanne/Notebook/Wet Lab

From 2009.igem.org

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[http://partsregistry.org/Part:BBa_I6007 Inverter TetR (BBa_I6007)]  was transformed again. And the new plasmid (created on July the 10th, [https://2009.igem.org/Team:EPF-Lausanne/Notebook/Wet_Lab#10.07.09 10.07.09]) LacI-RBS was transformed on DH5-alpha competent cells.
[http://partsregistry.org/Part:BBa_I6007 Inverter TetR (BBa_I6007)]  was transformed again. And the new plasmid (created on July the 10th, [https://2009.igem.org/Team:EPF-Lausanne/Notebook/Wet_Lab#10.07.09 10.07.09]) LacI-RBS was transformed on DH5-alpha competent cells.
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The purified  [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)] was concentrated, according to protocol cf. page 23 lab notebook.
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We failed in purifing our [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)], do it again tomorrow, beginning with the PCR, etc.
===14.07.09===
===14.07.09===

Revision as of 15:41, 13 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):

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 PCR, etc.

14.07.09

15.07.09

16.07.09

17.07.09

20.07.09

August