Team:KULeuven/18 July 2008

Wet Lab
Both protocols we tried yesterday failed to give any colonies on the ampicillin plates, even the pUC control plasmid failed (but this is probably due to a "bang" during electroporation). That's why we checked if there is any DNA in the paper spots at all. We punched out a part we did'nt need and measured the concentration with the nanodrop. This indicated that there was very little DNA and a lot of contamination present. The sample was also prepared with EB buffer instead of TE buffer, but this gave even worse results with the nanodrop. The sample was also loaded on a gel to see if there was DNA. We were all very dissapointed when we saw no plasmid after running the gel. The parts we need were ordered from IGEM HQ, we hope they'll arive soon.

And now for something completely different: the protocol to make competent cells was continued.

Modeling
We all had a tough day modeling today, even though everything seems to be parameterized now. We had a lot of technical problems with CellDesigner. Maarten had a lot of fun setting all the species and kinetics right in our model: cutting and pasting seems to be a big no. Because of this, the modeling has shifted a bit towards MatLab (aka Shmatlab) which seems to lack an undo button for some things.

Nick has been testing the filter and it's resistance to noise in CellDesigner. The output looked very good, signals of 1/5th of the maximum input and with a duration of 300 seconds were efficiently fitered out. During the meeting Inge suggested that we should take a look at the effect of the RiboKey lifetime on this filtering.

However, in the afternoon, a working model of the whole system minus the memory and the output has been constructed in CellDesigner. We have inspected some output which created suspicions towards the compatibility of the (filter-lactonase-celldeath)- with the (inverter-LuxI)-system. Even though the two systems work like a charm seperately, the complete simulation doesn't look so good at first sight. We suspect this has got something to do with quantities that don't match. We hope it might be solvable by changing copy numbers of the genes.

Simulating the memory is proving to be a problem as well. It reaches its stationary state (1) way to fast causing the 0-state to be virtually inexistant. In the simulations, tiny amounts of the activator seem to accumulate, finally causing the ultimate switch to the 1-state. We hope these tiny amounts (10E-8 -ish) are not biologically relevant, the bright side of this is that auto-activation of the memory should result in cell death, automatically filtering out the auto-activated cells if the input remains off.

Anyhow, we've got more than enough to keep us occupied :)

Remarks
We had our weekly meeting today. The report can be found here.