From 2011.igem.org

Week 17

From Monday the 10th of October to Sunday the 16th of October 2011

Monday

Strain construction

We're back to the lab for few more experiments, so we're building up again the strain we'll need for these experiments.

Transformations : PHL1414/pIG2, PHL1414/pIG6, PHL1414/pIG16, MC4100/p85, MC4100/pIG2, MC4100/pIG6
Note:There was less than 2µl of p85.
Dickeya strain A350 was transformed by electroporation with pIG6 and pIG2. A 5ml of LB liquid culture of 1 clone of both strain is started.
Do not forget that Dickeya doesn't like the cold, so keep the Petri dishes on the bench.

Tuesday

Strain construction

Transformations results:
MC4100/pIG6 --> 1 clone
MC4100/pIG2 --> 2 clones
MC4100/p85 --> 0 clone
PHL1414/pIG2 --> 0 clone
PHL1414/pIG16 --> 5 clones
A 5ml liquid culture of each clone obtened is started.
Extraction and digestion by E and P.
The gel electrophoresis shows that the clones of MC4100/pIG2, MC4100/pIG6, PHL1414/pI16, PHL1414/pIG6 and A350/pIG2 are ok. There is nothing on the digested A350/pIG6 track.

New test for the transformation of pIG20 (same as pIG2) in PHL1414.

Strain collection

S46 = A350/pIG6
S45 = A350/pIG2
The final concentration of glycerol is 40%. (500µl of culture + 500µl of Y80%)

Wednesday

Strain construction

Transformations results: PHL1414/pIG20 --> 3 clones
A 5ml liquid culture is started for two of them.

Adherence Test Preparation

Start of 5mL liquid cultures in M63G medium of PHL1414/piG6 (AmpR), PHL1414/piG2 (AmpR)
PHL1414/piG3 (AmpR), PHL1414/piG16 (AmpR), A350/piG6 (AmpR) and A350/piG2 (AmpR).
A350 = Erwinia strain

Thursday

Adherence Test

Start of three series (two replicates per series) of 24 well plates in M63G medium + Amp :

- PHL1414/piG6 without Co and with Co 10µM, 25µM, 50µM and 100µM (negative control)
and PHL1414/piG2 without Co and with Co 10µM, 25µM, 50µM, 100µM.

- PHL1414/piG6 and PHL1414/piG3 without Co and with Co 25µM (two negative controls)
and PHL1414/piG2 and PHL1414/piG16 without Co and with Co 25µM
--> In order to compare the adherence of our parts : pRcn-csgBAEFG and 18A-OmpR234

- A350/piG6 without Co and with Co 10µM, 25µM, 50µM and 100µM (negative control)
and A350/piG2 without Co and with Co 10µM, 25µM, 50µM, 100µM.
--> In order to test the effect of the part pRcn-csgBAEFG in another strain GRAM -

Incubation at 30°C for 48h.

Saturday

Strain construction

Transformation of p85 in NM522 which is an easier strain to transform than MC4100.
As there was no liquid left in the eppendorf tube from the plasmid collection, the tube is rinced by 5µl of steril water and the transformation is done with this.