EPF-Lausanne/4 September 2009

  

4 September 2009

Wet Lab

 * Glycerol stock for the readout 1
 * Miniprep on the 3 clones

PCR check + digestion assay:  Out of the 9 tubes for the miniprep (3x5mL for eachclone), 3 tubes of the same clone were red: Contamination? → maybe it is not the construct (altough we took these ones after check of the colony PCR of the previous day 03.09.09)

2 tests:
 * PCR of each miniprep clone
 * digestion assay with SpeI

Characterization


The plot shows that we still have trouble with the media... The only cells taht shows an induction are cells A1-A2, B1-B2, C1-C2, D1-D2, E1-E2, F1-F2. The only cells expressing RFP are RO2 #6 and #7, when cultivated in LB only (which contains Trp), that is only LB did show a "responsive pattern". This is strange because the gel showed that #6 and #7 did not have the construct wihle #8 had it.
 * → Maybe it is that there are all minimal and that the cells need more than 2h to make protein synthesis

A1-A2, B1-B2, C1-C2: (without Trp added) It shows a plateau after about 1h30 D1-D2, E1-E2, F1-F2: All our synthetic media had a flat linear increase in fluoresence over time, for an unknown reason.
 * → an explanation could be: in A1-A2, B1-B2, C1-C2, sone RFP is expressed because of the Trp in the medium. The cells are still in the growing phase, so the plateau is explained by the stationary phase of growing.
 * In D1-D2, E1-E2, F1-F2, there is some expression of RFP after 1h30.

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

CCL: The 3 media without Trp do not work.

But : we forgot to put ATC !!

People in the lab
Basile, Mélanie, Caroline

 