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. Evertime 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.

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).

Reculture

Made one new bottle of M9 min + AA + thiamine in 500 ml.

All clones (M9 & LB) have grown. M9 cultures have been re-done in 25 ml M9 + antibiotic. 750 ul of overnight re-inocculated.

Put in incubator at 37°C for 2 hours.

We got different OD after this time.

RBS, LacI-RFP #1,2, RO2 #4,5,10, RO1 #1,2,3.

We normalized the OD to 0.06 for all of them by adding the appropriate amount of fresh medium to each erlen (the calculation was done linear).

Characterisation

We took our cultures and did different conditions : -Trp, +Trp (0.5, 1, 1.5 where 1x is 2ul), -ATC, +ATC (0.5, 1, 1.5 where 1x is 1ul), +IPTG (1x = 5.5 ul), without anything.

From the start of the induction to putting the plate in the machine, it took about 20 minutes (so osome wells may already have been induced).

People in the lab

Mélanie, Caroline, Basile, Nicolas