EPF-Lausanne/10 October 2009

Wet Lab

All cultures have grown

IPTG stock solution

200 mM (for induction at 2 mM -> 100x) in 15 ml, so 0.714 g.

Setting the conditions for the culture:

Different conditions :

- + light / + IPTG / - TRP

- + light / - IPTG / - TRP

- - light / + IPTG / - TRP

- - light / - IPTG / - TRP

- - light / - IPTG / + TRP

Each condition for RO2.4 + BB1 /#3 DH5a LB and for RO1.1 + BB1/#1 DH5a LB.

Pre-prepared stock to aliquot : 3.5 ml of corresponding culture in 27 ml of new fresh LB (+ antibiotic). Flasks : +/- IPTG - trp, - IPTG + trp. Induction : TRP : 0.9 mg/ml, ITPG : 2 mM. Then we put the aliquot in eppendorf tubes (1ml).

Measurements : with abs/fluo on plate reader.

Each time a plate is prepared, the corresponding aliquot is used once only.

We put 200 ul in each well.

One plate served twice (2nd time the non-used wells were used).

qPCR experiment

Reinoculate 3 mL in 100 L.

For each mix, we put 900 ul of cell's solution and add TRP,Atc or LB depending on the condition.

RO1 : only (+100 ul of LB), + TRP, + TRP 2/3, +TRP 1/3.

RO2 : only (+100 ul of LB), + TRP, + TRP 2/3, + TRP 1/3, + ATC, + ATC 1/2, LB only.

TRP : 1x corresponds to 100ul of TRP, 2/3 to 67 ul of TRP + 33 ul of LB and 1/3 to 33 ul of TRP + 67 ul of LB.

     The initial stock of the TRP solution is 9mg/ml and the amount

ATC : 1x corresponds to 20 ul + 180 ul LB so 100 ng/uL, 1/2 to 50 ng/ul (10 ul of ATC added in 90 ul LB).

Total in each well for the measurements : 30 ul.

Results

=Kinetic experiment

Observe the degradation of RFP over time with the plate reader. Observation on RO2.4 + BB1 and RO1.1 + BB1.

For each clone, 2 conditions : + IPTG / + light, - IPTG / - light.

Transformation

Making RO2 + BB and RO1 + BB in JRG 465 / JRG 1046.

50 ul of cells :

RO2 + BB : 5 ul of the minipreped double transformants (containing both RO2.4 + BB1 /#3). Conc : 450 ng/ul.

RO1 + BB : 4 ul of RO1.1 at 154 ng/ul and 2.5 ul of BB1 at 250 ng/ul.

People in the lab

Christian, Heidi, Basile