EPF-Lausanne/16 October 2009

  

16 October 2009

Wet Lab
Miniprep of liquid cultures containing the plasmids with the possible (though unpure) mutated LovTap gene.

Digestion assay of LovTap in iGEM plasmid (the one we just cloned these last days): we digested it with EcoRI and PstI. In theory we should obtain the cut plasmid, and 3 bands at respectively about 150, 250 and 350 bp. The gel photo confirming that our LovTap is correct is here:



Prepared a new M9 medium to do further tests on RO1 and RO2.

Did an SDS-PAGE to test the level of expression of our LovTap biobrick with +/- IPTG added to the culture medium of the cells. Ran the gel, stained it, started destaining it. End of the protocol and photo were finished on Saturday.

Characterization of the RO2
Tried to test RO2 with different conditions. The measurements were performed in LB and M9 medium. For each medium, we tried to induce the RFP expression with Tryptophan or Anhydrous Tetracyclin. Two different solution of Tryptophan (with a final concentration of 0.9 mg/ml in the cell's solution) were tested: one solution from last week which was kept in fridge without light and a new one with a room temperature. The Anhydrous Tetracyclin was made the same way as on the 21st August (e.g. in a 50% EtOH solution as writen in the user's manual of the ATc we received).

Here are the results of the qPCR:









We can see that our experiments didn't worked, there is no response to Tryptophan as the curves are constant in the relative difference plot. Something unexpected happened, the response to the ATc shows a decrease of RFP instead of an increase. This difference is more expressed in the M9 medium.

In the M9 medium, the data seem to be inverted (cells in M9 only and +ATc). If this was the case, we would obtain the graph below of the relative difference which would be more logical.



In conclusion, our Readout 2 seems to have a problem:

Maybe the glycerol stock is too old => we will re-do a transformation of the RO2 from new plasmid.

Maybe the ATc in 50% EtOH provide a stress on cells, but it worked on 21st August and it doesn't explain why the cells don't responde to Tryptophan.

People in the lab
Tu, Gab, Basile, Christian

 