EPF-Lausanne/28 August 2009

Wet Lab

We did a glycerol stock out of the 3 LR2+GFP clones : LacI-RBS #1, #2 #4 + GFP (E02110).

Characterisation of RO2 + LacI-RBS

We used the reader in Sebastian's lab (qPCR machine).

We took 3 clones of RO2 (3 in M9, 3 in LB), and put 50 ul in each well. In 3 wells we add ATC (5 ul), in 3 other nothing, in the next 3 TRP (5 ul) and finally in the last 3 IPTG (5 ul, to induce LacI).

It was put for 2 hours at 37°C and 1 picture was taken each 3 minutes.

The results weren't concluding :

- All clones with ATC (no matter M9 or LB) had a "linear" progression.

- The clones (M9 and LB) with nothing and TRP didn't make any difference (linear half way then growing a little bit exponentially).

- LacI-RBS-GFP clones didn't seem to react to the IPTG induction (same pattern as those which hadn't been induced).

Analysis of the results :

1. LacI-RBS-GFP : it is strange, we will have to redo it with a positive control for GFP and one for the LacI promoter.

2. RO2 # 4/5/10 : it is actually quite sensible that we got this if we consider that LB and M9 medium are saturated with TRY. Some overnight culture are already red, without any induction. It also explains why ATC induction doesn't induce anything, as there isn't any TetR protein (saturated with W).

We definitely need to have a medium with no TRY to test and caracterize our RO2. Morever, we should also add positive control to our plates with RFP (inducible) to see what normal fluorescence is. We could make an artificial media including all amino acids but no TRY.

TrpO

On the last gel it was looking fluffy so we aren't sure if the reaction has worked (considering that the ligation and the transformation didn't work).

We did a PCR on our TrpO (obtained by Klenow reaction). This was to see if maybe we had too much product that haven't been made finish by the enzyme, like no re-formation of the phosphodiester aduct by the klenow etc. For the PCR, we used the normal Taq platinium protocol and as primers we used iGEM standard as forward and Trp operon as reverse primer.

We ran a gel, and according to this, we can say that probably we have the right sequence. It is still fluffy, but they are small fragments, which is concordant with the "fluffiness" of the 100 bp ladder.

People in the lab

Basile, Rafael, Nicolas