EPF-Lausanne/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:

• 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.

CCL: The 3 media without Trp do not work.

But : we forgot to put ATC !!

People in the lab

Basile, Mélanie, Caroline