Team:EPF-Lausanne/LOVTAP Results

Parts

The biobrick parts (inducible LovTAP, RO1 and RO2) have been produced. We are currently sequencing them to confirm that there are no errors in the insert. You can have more information about these parts here

Characterization of RO1 and RO2

We characterized RO1 and RO2 by cultivating cells containing one of the read-out systems under different conditions :

- With TRP

- With ATC

- With both

- Without anything

And at different concentrations.

We had some troubles with the media, as we usually cultivated the cells in LB, but it contains Trp, and so it pertubated the results. Therefore we had to find a medium in which there was no Trp but on which cells could grow. We did a lot of different tests and we finally settled on a medium containing M9/minimal, amino acids and thiamine. At the beginning we also compared the results between growth on LB or M9 to see the difference.

Here are the final graphics that characterize our two read-out systems. More details can be found in the notebook section.

For RO1 :

RO1 final plot

So we can see that there is a decrease in RFP expression when TRP is added. Therefore, we can admit that our Trp op is functional. We considered the characterization for this part done.

For RO2 :

This experiment was done in LB medium. All cells were normalized to an OD's of 0.2 before adding inducers which were put into the liquid culture just before the measurements. The concentration of Tryptophan was 0.9 mg/ml and the concentration of Anhydrous Tetracyclin was of 50 ug/ml.

Here are the result from the machine (qPCR).

Clone 4 plot

Here is an average signal for each condition as we had 4 measurements for each state

Clone 4 plot Mean

A last picture shows the relative difference between the signal from cells with Trp or ATC and the signal from the cells' background fluorescence.

Clone 4 plot RelDiff

This confirms that our construct responds both to Trp and ATC and shows the characteristics we expected.Therefore, we can suppose that both the Trp op and the Tet op are functional and that we can use this part in order to see if the LovTAP is functional.

Characterization of the entire system

The first result we obtained after an illumination of the entire system was the following :

RO2-BB#1 plot

We can see that our system mimicks the response with TRP, which is exactly what we wanted.

We first did one static measurement of fluorescence. For the + light condition we exposed the cells to the light for 2 hours.

Then we wanted to see the time course of the induction : when is the RFP expressed, how etc.

It appears that this actually happens during the first hour.