EPF-Lausanne/10 September 2009

Wet Lab

Colony PCR

We did a colony PCR of the 3 BB / Chl (A, B, C) that have grown (after an overnight + 1 day culture on a plate). It is strange they took so long to grow, so we hope it is not a contamination.

We took a couple of clones on each plate and used the Taq platinium protocol + colony file. We will run a gel to check if the clones are what we wanted.

Results of the culture

All the tubes grew (RO2 / RO1 / double transf.). The medium was only M9 (M9 + AA + thiamine) and not LB this time.

- RO1 #1 (red), #2 (red) which is very nice as it is in M9 (-TRP).

- RO2 #4 (white), #5 (redish), #10 (red). The white one is a good result, but the other should be white as well.

- RO1 + BB #2/3 (white)

- RO1 + BB #3/3 (white)

- RO1 + BB #1/1 (white)

- We have noticed for a few days that RO1#3 doesn't work, there might be a mutation as it never gives the right results.

- The RO1 should be red ! So it could be that the LovTAP is working (upon illumination, LovTAP binds its operator Trp and inhibits TrpO and thus RFP).

- RO2 + BB #8 (RO2 #10 + BB6, clone #8) -> slightly redish

- RO2 + BB #3 (RO2 #4 + BB1, clone #3) -> slightly redish

All have been re-inocculated (500 ul) in 25 ml of fresh M9 + antibiotic, and put in incubator at 37°C at 9.15 am.

Glycerol stocks

The 4 "yale" strains have grown (hopefully with no contamination). Note : the strain JW4356 11110 grew in Kana as well -> it's kana resistant.

The other : JW4356 11110 (LB/no antibiotics), JRG465 4456 and JRG1046 5992 all grew in SOB. We will make them competent tonight (100 ml at 3 p.m. at 18°C) and wait until OD = 0.6.

At 2p.m. : the 5 double transformants have been induced at 1mM of IPTG (stock solution : 0.238 g IPTG in 10ml MG). Put at 37°C.

Gel

50 ml, 1%, to check the construction BB/Chl (P5B4C5).

Result : we should see a band at about 1100 bp but we don't. So apparently the vector just religated on itself. We will have to try another colony PCR tomorrow with a positive and a negative control this time !

Making chemically competent Trp K.O. strains

Did 500 ml of SOB -> pH = 7.5, sterilized.

4 erlen of 1000 ml, in each 100 ml of SOB and 600 ul of Kana in one. Then added 1,5 ml of the overnight culture to the corresponding erlen'. At 18°C (220 rpm) from about 3 p.m. We will let them overnight until OD 0.6.

Characterization

We did another characterization, but this time with the LovTAP construct. To do : redo the experiment by culturing the bacteria in the dark. RO1 without IPTG and without light. 2 plates : control with and withoud light. The idea could be to load the wells in a dark room, with IR light ?

We used the following constructs in this experiment : RO2#5, RO1#2, RO1# + BB5 #3, RO1#1 + BB1#1, RO2 + BB#3, RO2 + BB #8, RO1#2 + BB5 #3, RO1#1 +BB1 #1, RO2#4 + BB+ #3, RO2#10 +BB6 #8, RO1#2, RO2#4, RO2#10.

The conditions were the following :

RO1-BB : without light : +TRP / -TRP. With light : +IPTG / -ITPG.

RO2-BB : without light : +TRP / -TRP. With light : +IPTG / -IPTG.

People in the lab

Mélanie, Caroline, Basile, Nicolas