Team:Cambridge/Quiescence Notes

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Revision as of 11:20, 16 July 2010

• [http://mic.sgmjournals.org/cgi/reprint/155/8/2676.pdf The role of FIS in the Rcd checkpoint and stable maintenance of plasmid ColE1] ie good as the toggle control thingy
• [http://www3.interscience.wiley.com/cgi-bin/fulltext/119127730/PDFSTART Timing, self-control and a sense of direction are the secrets of multicopy plasmid stability]
• [http://aem.asm.org/cgi/reprint/65/6/2710.pdf The quiescent-cell expression system for protein synthesis in Escherichia coli]
• [http://apps.isiknowledge.com/full_record.do?product=UA&search_mode=GeneralSearch&qid=12&SID=W11BA3C7bKeHC5PbfoE&page=1&doc=1&colname=WOS ColE1 multimer formation triggers inhibition of Escherichia coli cell division.]
• [http://books.google.com/books?hl=en&lr=&id=OKn60-HZisUC&oi=fnd&pg=PA19&dq=hn-s+nucleoid+bacillus&ots=rstjtFpQ2Z&sig=15uulXPnHz-yyXMvMKRrTXcLk3U#v=onepage&q=h-ns&f=false book about bac genomes]
• [http://www.faqs.org/patents/app/20090004700 D. Summers' Patent Application for Chem. Induction of Quiescence in Bacteria]

Literally no documentation of h-ns and rcd in bacillus.

If we do quiescence, will have to be in e coli with bacillus as a side project. Have to get over IP issues with ucam + e coli. Implementation in ecoli will be fairly easy as it is a working system - all we need to do is brick it, and get it working.

Input from James

• Has to be done in E.Coli, will not work in non Coliform bacteria such as Bacilli.
• Switch on cell division would be very useful, however there are IP issues which must be discussed with David Summers.
• Another constraint is that liquid broth not agar must be used.
• Nobody has looked at it from a microfluidics point of view.
• When done before, the lambda phage takes 3 hours to initiate quiescence after the temperature change.
• The Lambda phage is the most stable to have an off switch, it does not express itself when needed (?)
• The RNA has a very long half life, it would be hard to strip away the system and put it under a new promotor, but, if possible would be extremely useful

Structure

A new approach to making a quiescence switch would be to control the functionality of the folded RNA instead of its transcription, which proved to be problematic. Some data on the structure of different Rcd can be found here:

• [http://mic.sgmjournals.org/cgi/reprint/145/8/2135 1999 article by Sharpe et al. Summers being one of the al]

Gos really likes the idea. He suggested using a self-splicing RNA switch that is active in the absence of a ligand. Thus the bacteria would grow in the lab or a vat in the presence of the ligand but would stop growing without dying, as soon as the ligand is removed. Thus if your bacteria escaped into the wild they would stop growing very quickly as the ligand is diluted. The project would involve synthesising lots of different constructs (which are all mercifully short) and testing their functionality and sensitivity. The result would be a fairly short, but quite versatile BioBrick. Gos recommended talking to Jim H and David Summers about whether a self-splicing structure in this functional RNA can work.

We've got The Aptamer Handbook from the Central Science Library, it describes about three billion compounds you can make aptamers bind to, and has a whole section on applications, but unfortunately doesn't seem to mention self-splicing. -.-

Paper on ribozymes, including sweet self-splicing ones:

[http://lim.fcien.edu.uy/tallerbm/pdf/2.%20Serganov%202007%20Nature%20Rev%20Gen.pdf Swish Ribozyme review]