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Team:KULeuven/18 July 2008
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*Both protocols we tried yesterday failed to give colonies on the ampicillin plates, even the pUC control plasmid (but this is probably due to a "bang" during elektroporation) | *Both protocols we tried yesterday failed to give colonies on the ampicillin plates, even the pUC control plasmid (but this is probably due to a "bang" during elektroporation) | ||
| - | *That's why we checked if there's any DNA on the paper spots at all. We punched a part we did'nt need and measured the sample with the nanodrop. This indicated that there was very little DNA and a big part of the peak would be probably contamination. The sample was also prepared with BE buffer instead of TE buffer, this gave even worse results. The sample was also loaded on a gel to see if there was DNA. We were very dissapointed when we saw no plasmid after running the gel. The parts we need were ordered from IGEM HQ. | + | *That's why we checked if there's any DNA on the paper spots at all. We punched a part we did'nt need and measured the sample with the nanodrop. This indicated that there was very little DNA and a big part of the peak would be probably contamination. The sample was also prepared with BE buffer instead of TE buffer, this gave even worse results. 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. |
*The protocol to make competent cells was continued. | *The protocol to make competent cells was continued. | ||
Revision as of 16:52, 18 July 2008
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Contents |
Lab Work
Wet Lab
- Both protocols we tried yesterday failed to give colonies on the ampicillin plates, even the pUC control plasmid (but this is probably due to a "bang" during elektroporation)
- That's why we checked if there's any DNA on the paper spots at all. We punched a part we did'nt need and measured the sample with the nanodrop. This indicated that there was very little DNA and a big part of the peak would be probably contamination. The sample was also prepared with BE buffer instead of TE buffer, this gave even worse results. 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.
- The protocol to make competent cells was continued.
Dry Lab
We had our first weekly meeting today.
About the modeling
Modeling
We all had a tough day modeling today, even though everything seems to be parameterized now. 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 relevent, 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 :)
