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):

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)

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 (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 transformation of the ligation product (with Amp and not Chl antibiotics this time !!) and Term.

17.07.09

The plates and tubes from yesterday transformation weren't very concluant : almost no colony on the plates (actually we saw 3 of them but this might be contamination) and for the tubes there were almost no bacteria. We decided to do the miniprep with it anyways, to see what it gives :

Miniprep of LovTAP-Term, CT negative of the ligation and Term (that grew very well).

PCR of the miniprep products.

Gel electrophoresis of the PCR products and also of the plasmid of LovTAP-Term, CT neg and Term. We can see on the gel that there was actually no DNA on the LovTAP-Term and CT negative tubes.

In parallel, we began all over again, by taking Term from the kit. We used a different method, as the other one seems not to be working (the Term plasmid seems not to accept LovTAP in it). So this time we used the iGEM protocol to do the digestion. As the P site is in LovTAP, which is the upstream part, it is not a problem.

We did the digestion, ligation and transformation of LovTAP-Term and this time we really hope this will work.

18.07.09

Result of the transformation: nothing grew.

20.07.09

As the transformation of Saturday didn't work, we think that the plasmid Term BBa_B0010 has a problem. We tried to begin all over with 4 other Terminator from the registery.

We first did an extraction of 4 new Term plasmid from kit plate. Then we did a transformation of these 4 Term + a transf. with death cassette (with the special protocole for One Shot ccdB survival cells).

At the same time, we did a PCR with the 4 plasmids from the kit + LovTAP. We purified these PCR products, in the idea to do a digestion and then a ligation with them.

In parallel, we ran a gel with the Term plasmids, but there were no bands on the gel, so we didn't do the digestion. We will wait to have the transformation products to do that.

We also prepared LB-plates : Amp-Kana, Amp-Kana-Chl and Kana-Chl.

21.07.09

The transformation of the Term plasmids worked, so we did glycerol stocks of the 4 new Term and the death cassette.

Then miniprep of the 5 transformation products.

Digestion following the iGEM protocol : we cut with X and P Term, E and P the backbone and we take a LovTAP that was digested Friday with E and S.

Running of a gel to check the digestion products. Actually it seems the digestion didn't work. We did a second gel, by loading more product, and we clearly got the same result.

As the ligation this way seems not to work (either the enzymes for the digestion aren't working, either there is another problem in the ligation), we decided to try another way to do it : by doing a 2 step PCR. We designed and ordered the primers, now we just have to wait for them to go on in our cloning of LovTAP. Have a look at the cloning strategy to see how we designed the primers.

The idea of 2 step PCR : Media:2step pcr‎.pdf

22.07.09

We sent the miniprep product of LacI-RBS to sequencing. With all the problems we have with LovTAP-Term, we are really not sure that LacI-RBS really ligated well, so we want to have the sequence to check.

23.07.09

Plates with different bacteria concentrations were made to study which is the best concentration (amount) of bacteria we might let grow on plates.

Results of the sequencing of LacI-RBS : none... actually they weren't able to sequence it, it seems not to be pure enough. So we will do the ligation again today, including many control steps this time !

24.07.09

We found a Term plasmid that last year team used, and that worked : BBa-B0015.

We did 3 ligations in parallel :

LovTAP-Term with iGEM protocol and using the plasmid BBa-B0015.

LovTAP-Term using the Term digested last year with EX. (The idea is to insert LovTAP in the Term directly)

LacI-RBS with iGEM protocol again.

So we first did the digestion :

1. LovTAP cut with E and S, Term plasmid cut with X and P, the backbone cut with E and P.

2. LovTAP cut with E and S. We will use the Term from last year, already digested.

3. LacI with E and S, RBS with X and P and the backbone with E and P.

Next we ran a gel to check the digestion products.

We did the 3 ligations in parallel.

At this time we had the results of the gel, and actually the digestion didn't work. The backbones were digested but not the rest. There were no bands at all for the LovTAP and the others weren't digested. We will begin all over but using another protocol than iGEM's for the digestion

27.07.09

LOVTAP gene have been amplified again and runned on a gel to be sure to have the correct insert.

BBa_B0015 was digested, runned on a gel (which displayed the correct band) and extracted with the gel extraction kit, this step didn't worked well.

We already had a digested BBa_B0015

28.07.09

29.07.09

30.07.09

31.07.09

August