EPF-Lausanne/8 September 2009

Wet Lab

Cultures

Results:

RO1 has grown in LB and M9

RO2 has grown in LB but not in M9

RO2+BB #1,4 did not grow

RO2+BB #3 has grown in LB but not in M9

-> problem with our glycerol stock (RO2 + BB didn't grow in LB !!). We did some plates of the cultures that grew.

For safety we did some plates with the glycerol stocks of RO2 + BB 1,4,8. We also did some plates with the cultures that grew (RO1 1,2,3, RO2 4,5,10, RO2 + BB 3).

Characterization

As RO1 grew in both media, we put it in 25mL medium with Amp. We did also with RO2 (LB), and RO2+BB (LB), and LacI-RFP.

For the characterisation, we did different combinations : -TRP, -TRP -ATC, +TRP -ATC, +TRP +ATC. Every time for all the clones in M9 and then in LB.

TRP : LB : dilution 50x so 1ul, M9 : dilution 25x so 2 ul. We took the stock solution 4,250 g/L.

ATC : 5x dilution so 10 ul from the 500 ng/ml stock solution.

The aim was to get used to the machine and to see the differences between LB or M9.

Here are the results of the characterization :

RO1#1 + TRP in LB There is already some TRP in LB so it shouldn't change a lot.

RO1#1 without TRP in LB This doesn't mean a lot as there is already some TRP in LB but we can try to see a difference with RO1#1 + TRP in LB.

RO1#2 + TRP in LB

RO1#2 without TRP in LB

RO1#3 + TRP in LB The clone 3 doesn't seem to be functional.

RO1#3 without TRP in LB

RO1#3 + TRP in M9

RO1#3 without TRP in M9

RO2 + TRP + ATC

RO2 + TRP - ATC

RO2 without TRP + ATC

RO2 without TRP and without ATC

Results of the transformation

They all grew : RO1#2 + BB5, RO1#1 + BB1, RO1#3 + BB3.

So we did a colony PCR to be sure that we have the double transformation, using the Taq platinium protocole.

We picked 8 clones of RO1#2 + BB5, 8 clones of RO1#1 + BB1 and 5 clones of RO1#3 + BB3.

We ran a gel to check if it has the two plasmids : for the clones that worked, we should see two bands. We ran a gel : we should get two fragments. We can see that all our clones worked (had the two constructs), except one : clone 5 for RO1#3 + BB3.

Transformation

Then we did a transformation of the ligation products : BB1, BB3, BB5, BB6 in a chlorophenical receptor, on Chl plates.

Culturing

LB : RO2 + BB (1,3,4,8), LacI-RFP (#1,2), BB/Chl (results of the gel : 4 clones). M9 : LacI-RFP (2 clones), RBS BBa_B0030 (control for the fluo. of the cells), RO2 (4,5,10), RO1 (1,2,3).

People in the lab

Mélanie, Caroline, Basile, Nicolas