Team:KULeuven/24 July 2008

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Lab Work

Wet Lab

Stefanie and Hanne made a glycerol stock of all the parts we received from the iGEM headquarters. Everything is now stored in two -80°C freezers. You can find the cells with the parts in box S&P-F7. You can look up in the cmpg database where you should search for a specific part.

In the afternoon we started the protocol to make the desired E.coli strain by transducing the EnvZ-mutation from V1012 to MC1061 cells. Therefore we made a liquid culture of the donor and acceptor cells. Phage P1 was incubated with the acceptor. Tomorrow we'll continue the proces and bring phage P1 and the acceptor cells (MC1061) together, this should result in the right cells.

Because the concentrations of the isolated plasmids were low yesterday, Elke, Jan, Andim and Nathalie did it all over again. However, this time we tried to obtain higher concentrations of plasmid DNA. Normally, this will improve the success rate of the digests. We did this for the following BioBricks:

BioBrick	Sample 1: Concentration	Sample 2: Concentration
B0032	114.8 ng/µl	/
B0034	73.8 ng/µl	95.1 ng/µl
E0022	86.4 ng/µl	/
I714891	140.9 ng/µl	/
J23100	345.0 ng/µl	176.1 ng/µl
M30109	54.3 ng/µl	/
R0084	66.9 ng/µl	60.9 ng/µl
B0015	112.3 ng/µl	130.3 ng/µl
C0040	177.1 ng/µl	161.7 ng/µl

Then the plasmids were digested with the appropriate restriction enzymes (EcoR I, Spe I, Xba I). In order to see whether the digestion worked, we put the samples on an agarose gel. The plasmids with BioBricks I714891 and B0015 were not cut properly by the enzymes. The other parts were sliced out of the gel and are ready to be purified and ligated.

BioBrick	Restriction enzymes	length desired fragment	success rate digestion
B0032	EcoR I, Xba I	2077	probably
B0034	EcoR I, Xba I	2077	probably
E0022	EcoR I, Spe I	783	OK
I714891	EcoR I, Spe I	757	failed
J23100	EcoR I, Spe I	51	OK
R0084	EcoR I, Spe I	129	OK
B0015	EcoR I, Xba I	2193	failed
C0040	EcoR I, Spe I	681	OK

Dry Lab

Primers

Primers for the construction of T7 polymerase with UmuD tag were written out.

Modeling

Pulse generator has been reviewed, the parameters have been checked but there are issues... The pulse generator doesn't work (pure physically): when a step signal of T7 is applied cI is produced, which will then repress its transcription. Of course, this will develop to a steady state with a cI level equal to a non zero value. A zero cI value would result in zero repression, thereby restarting the transcription of the cI gene and start producing cI again anyway. Because the cI value remains non zero, so will the lactonase production, and so there will always be a constant amount of lactonase present. Question is: how much will this concentration affect the system considering the case of no light present? No light present means HSL production, which should bind to LuxR, but instead will react with lactonase to an hydroxy-acid. If the concentration of lactonase is considerable, and remains somewhat steady, cell death would never occur. Interesting link to a pulse generator system that seems to work: Pulse Generator Keasling Lab

The lactonase amount does receed to 0 when the filter has 0 output, when the inverter gives high input, but this could cause delay to cell death, due to the slow degradation of lactonase.

The memory system has been reviewed as well. The old system seemed unsuccesful, a new system has been found proven to work. There are however not enough repressors available to implement this system without having interference with other parts. ...

Wiki has been updated a bit more. SBMLSqueezer was used to produce pdf files with the celldesigner reactions, species and parameters. Biobricks pictures were added and some other stuff.

Remarks

iGEM gave us plasmids in cells. Which cells?

Quote of the day

GAS!