Team:Cambridge/Quiescence

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(The aptamer-controlled-ribozyme approach)
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==The aptamer-controlled-ribozyme approach==
==The aptamer-controlled-ribozyme approach==
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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. Ideally the bacteria would grow in the lab or a vat in the presence of a 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.  
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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. Ideally the bacteria would grow in the lab or a vat in the presence of a ligand but would stop growing without dying, as soon as the ligand is removed. Thus if the engineered bacteria escaped into the wild they would stop growing very quickly as the ligand is diluted.  
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We hope to achieve this by rationally designing a gene for an RNA that folds into a non-functional shape. The structure will include a hammerhead ribozyme domain that will hopefully cleave itself in vivo to release the functional Rcd RNA.
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[[Image:hammerhead_pre|250|left|thumb|3D-prediction of Hammerhead ribozyme before cleavage]] [[Image:hammerhead_post|250|right|thumb|3D-prediction of Hammerhead ribozyme after cleavage]]
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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.
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.
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According to mFold and CLC workbench, [https://2010.igem.org/Team:Cambridgee/Quiescence/Sequences the sequence we designed] folds into a non-functional form, as expected. The structure includes a hammerhead ribozyme domain that will hopefully cleave itself in vivo to release the functional Rcd RNA. This sequence does not include the aptamer yet.  
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According to mFold and CLC workbench, [https://2010.igem.org/Team:Cambridgee/Quiescence/Sequences the sequence we designed] folds into a non-functional form, as expected. This sequence does not include the aptamer yet.  
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713350/ Smolke et al. 2009] gives a nice review of aptamer activated hammerhead ribozymes and other functional RNA structures. More information on ribozymes (self- or trans-cleaving) can be found in [http://lim.fcien.edu.uy/tallerbm/pdf/2.%20Serganov%202007%20Nature%20Rev%20Gen.pdf Serganov and Patel 2007].  
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713350/ Smolke et al. 2009] gives a nice review of aptamer activated hammerhead ribozymes and other functional RNA structures. More information on ribozymes (self- or trans-cleaving) can be found in [http://lim.fcien.edu.uy/tallerbm/pdf/2.%20Serganov%202007%20Nature%20Rev%20Gen.pdf Serganov and Patel 2007].  

Revision as of 10:42, 28 July 2010