Team:KULeuven/6 August 2008

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Lab Work

Wet Lab

The electrocompetent cells were tested with pUC and we had a lot of colonies so these are working great.
Ligation products were transformed to these electrocompetent cells (E0022+B0015, J23109+J23032, I712074+J23032, GFP+B0015, GFP+pSB1A2, C0062+B0015, pUC as a control).
We made digest of J23032, F1610, J23022, B0015.
Miniprep colonies of the ligation.
The three punched parts of the memory gave a few colonies. One of them did not give any colonies at all. The colonies we had were plated on new plates.

Dry Lab

Check possibility of hybrid promoter to connect memory and timer (LuxI). This is to replace the antisense LuxI. Promotor would look something like this:
Operator_{HSL_LuxR} --- -35 box --- Operator_{CII P22} --- -10 box

New modeling of the antisense LuxI and its target LuxI mRNA shows that this leaky repression is not a problem for the system. Still looking at the composite promoter though.

Hybrid promoter has been made! [http://partsregistry.org/Part:BBa_K145150 Hooray for BBa_K145150!] It should be activated by HSL-LuxR and repressed by P22 C2 (which is produced when the memory is OFF, hereby repressing LuxI synthesis) Check tomorrow how to best obtain the sequence.

Literature
[http://jb.asm.org/cgi/content/full/190/13/4392?view=long&pmid=18083819 Lux regulatory region (pLux)]

[http://www.jbc.org/cgi/content/full/272/3/1646/f1 P22 C2 repressor operators]

PS: P22 C2 is homologous to lambda CI while P22 C1 is homologous to lambda CII

Modeling

The antisense part has been remodeled to more accurately show its leakiness. This however appears to be not a problem at all. Plus we can always replace it with the hybrid promoter.

Started work on the sensitivity analyses. So far so good as it would appear.

Further work on the memory because we got some new parameters. The switching has become somewhat trickier now.

Wiki

Dr. Coli is everywhere, even at the URL.

Remarks

Discovery of the day. Two people beat us to the punch: [http://www.diagnosticinformationsystem.com/bios.html 1] and [http://www.sscofcny.com/doctors/coli.htm 2]

Sindsdien hangt uw haar slap -Maarten