Team:Cambridge/Quiescence Notes

• The role of FIS in the Rcd checkpoint and stable maintenance of plasmid ColE1 ie good as the toggle control thingy
• Timing, self-control and a sense of direction are the secrets of multicopy plasmid stability
• The quiescent-cell expression system for protein synthesis in Escherichia coli
• ColE1 multimer formation triggers inhibition of Escherichia coli cell division.
• book about bac genomes
• 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:

• 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: Ribozymes ftw

Another very nice review on RNA parts and their functions. This article was one of the main sources for the talk we had on hammerhead+aptamer switches. Smolke et al. 2009

Papers

• Paper with diagram showing cut site: http://sage.ucsc.edu/scottlab/reprints/2006_Scott_Cell_pdf/2006_Scott_Cell.pdf

• 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:

1999 article by Sharpe et al. Summers being one of the al

• Paper on ribozymes, including sweet self-splicing ones:

Ribozymes ftw

• Another very nice review on RNA parts and their functions. This article was one of the main sources for the talk we had on hammerhead+aptamer switches.

Smolke et al. 2009

We do not necessarily have to use hammerhead RNA. There are other self-cleaving RNA structures that have been identified, as described in this paper. We could also look into using the aptamer as a direct means of changing shape and functionality of the RNA. That could end up giving us a less digital output and might be difficult to implement, as the Rcd RNA is a very stable structure.

Tang and Breaker 2000 on self-cleaving ribozymes

Sequences

Hammerhead ribozyme?: GGGAGCCCCGCUGAUGAGGUCGGGGAGACCGAAAGGGACUUCGGUCCCUACGGGGCUCCC

Rcd: AGGCGCGAUCGCGGCAGUUUUUCGGGUGGUUUGUUGCCAUUUUUACCUGUCUGCUGCCGUGAUCGCGCUGAACGCGUUUUAGCGGUGCCUACAAU Rcdv2AGGCGCGAUCGCGGCAGUUUUUCGGGUGGUUUGUUGCAUUUUUACCUGUCUGCUGCCGUGAUCGCGCUGAACGCGUUUUAGCGGUGCCUACAAU

Hammerhead sequence PDB: GGGAGCCCUGUCACCGGAUGUGCUUUCCGGUCUGAUGAGUCCGUGAGGACAAAACAGGGCUCCCGAAUU

THERE'S A APTAMER FOR CAFFEINE!!

Unfortunately it is massively insensitive. According to the aptamer handbook it (characterised by Jenison et al. 1994) has an equilibrium constant of 3500µM, at this concentration it has weak cytotoxic effects. There might be more sensitive aptamers for caffeine out there. Caffeine is very closely related to theophylline (which just has a -CH3 instead of a H), the aptamer for that has a Kd of 0.1 µM and serves as a standard example for aptamers. Its also found in tea. It might be less flashy than having caffeine responding cells, but it might be easier to make them tea-addicts. We're a British team after all? Definitely possible to spin the tea-angle, BUT, I think the 3500µM figure is for the theophylline aptamer's binding to caffeine. I now can't find a specific caffeine aptamer, but we might be able to find/make one? But theophylline is probably place to start..

Interesting in general + newer caffeine aptamer?

Tool for reverse complement

Doing it without a cleaving ribozyme?

http://gcat.davidson.edu/GcatWiki/index.php/Antiswitches