Team:KULeuven/13 August 2008

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  Input Output Filter InverTimer Reset Cell Death Memory

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Lab Work

Wet Lab

• We tested the ligations (using PCR and gel electrophoresis) of the following parts: J23109 + J23032, I712074 + J23032, K124015 + B0015, R0084 + J23022, R0062 + B0032 and C0012+ B0015. They seemed to have ligated properly. After that, we digested these ligations for the next step in the ligation process.
• We MiniPrepped the following parts: C0040 + B0015, C0060 + B0015 and J23100 + B0032. They will be tested tomorrow.
• We continued to make the T7 polymerase with UmuD tag. Therefore we used PCR in two steps. The first step worked, but the second one didn't. There might be something wrong with the primer (it was a very long one).

Dry Lab

Modeling

We seem to be on a streak this week as we finally solved the T7 issue as T7 transcription rates have finally been estimated realistically. Things are looking up again in this haunted week :)

Later however we encountered some weird stuff while simulating the entire system. Even without any light, the HSL-LuxR complex would still build up (extremely slow though) and would ultimately kill our cell. There appears to be a tiny flux of HSL through a tiny background amount of LuxI. The HSL produced quickly encounters LuxR transcription factors to which it binds. This binding protects the HSL from any background lactonase here (which has a much higher dissociation constant). When HSL does dissociate from the high affinity LuxR, either through degradation of the protein or through regular dissociation it will quickly be taken up again by another LuxR molecule because they're constantly produced due to the constitutive promoter. We solved this issue as well by putting the LuxR under a P22 c2 repressable promoter (= output of memory in OFF state)

At the end of the day we decided to try and put LuxR together with ccdB under control of the hybrid P22 c2 - LuxR promoter tomorrow. We'll see how the system behaves. Exciting ... ;)

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