EPF-Lausanne/4 September 2009

Wet Lab

Characterization

The plot shows that we still have trouble with the media... Only LB did show a "responsive pattern". All our synthetic media had a flat linear increase in fluoresence over time, for an unknown reason.
-> Maybe it is that there are all minimal and that the cells need more than 2h to make protein synthesis

For this test we actually did a mistake while picking the clones for the culture and we picked 3 out of which 2 didn't have the construct -confirmed by colony PCR- (therefore only the RBS-RFP-term).
-> only the 2 "wrong" clones showed a response! The one that actually had the readout 2 construct had the same fluorescent pattern as all the synthetic media pattern.

The 2 "wrong" clones had the following graph:

• without Trp: net linear increase of fluorescence for 1h30 then sudden change and the fluorescent slope went flat for the last 30 min.
• with trp: linear increase of fluorescence for the 2h.

The difference is then after the 1h30 where the ones induced with trp still had an increase in fluorescence while the other had not.

> As we got our readout1 (confirmed by PCR on miniprep + digestion assay), we > will do the following test on Monday: all in LB (while waiting for a better > option) > -Our 3 "right" clones of readout 2, induced by trp, Atc and nothing > -Our 3 readout 1 (confirmed) induced by trp and nothing > -A control of the "evolution of fluorescence over time" using the LacI > inducible RFP from the registry that we have. This, at least, we know won't > have any interferences.

People in the lab

Basile, Mélanie, Caroline