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).

Results of the plate reader

Data with only fluorescence expression, the unit is 15 min and at the point 1 is the first measurement at t=0min.

RO2 BB1 Fluo

Data with an OD normalization, the unit is 15 min.

RO2 BB1 Fluo OD

We conclude that at this time the experiment didn't work. The cells exposed to the light expresse less RFP instead of more!! We had a problem somewhere. We used ependorf during the light exposition, maybe the cells need more air to grow and work at optimum.

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

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 of qPCR

All graphs shows the average signal over 8 differents measurements of each condition.

RO1 :

RO1 FluoCurveAbs

The relative difference between the solution with inducer and without

RO1 FluoCurveRel

RO2 :

RO2 FluoCurveAbs

The relative difference between the solution with inducers and without

RO2 FluoCurveRel

As we can see on this graphs, the experiment didn't work as we expected like on 21 August 2009. The two Readout systems didn't respond neither to TRP nor to ATc. Maybe we should have keeped the reaction for more than 2 hours in the qPCR or we should have shaked more the mix before loading plate's wells.

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