IGEM:IMPERIAL/2008/New/Cloning Strategy

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</td><td align="center" width="17%" valign="bottom"><ul id="sddm"><a href="http://2008.igem.org/Team:Imperial_College/Notebook"> Notebook </a></ul> </td><td align="center" width="17%" valign="bottom"><ul id="sddm"><a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Team"> Our Team </a></ul> </td></tr></table></html>

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Cloning Strategy

The Imperial iGEM 2008 team faces the daunting task of working with a chassis that has been rarely used - and never characterised - in the competition so far. While the subtilis chassis offers us many advantages, working from the ground up like this also presents many challenges.

Our cloning strategy, for instance, is highly complex. In order to building increasingly complicated constructs for our final product, we need to build, test and characterise all the parts and devices leading up to the final systems! The diagram below shows the "critical pathway" for our cloning strategy and as you can see, there are a huge number of closely-linked steps..



A summary of constructs that should be produced by the end of each phase for testing is shown below:

Phase 1

Testing and characterisation of constitutive promoters. We will test 4 combinations of 2 promoters and 2 RBSs to characterise them. Antibiotic cassette is placed first on the construct, so that any readthrough from native transcriptase simply boosts production of antibiotic.

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase1.png"></html>

Phase 2

Testing and characterisation of inducible promoters; those marked with a 'c' are chemically-inducible and those marked with an 'l' are light-inducible. RFP is used instead of GFP to check output as ytvA responds to blue light - GFP may cause positive feedback. 'Rep' gene signifies a repressor for the chemically-inducible promoter to stop leaky expression.

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2A.png"> <img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase2B.png"></html>

Phase 3

Testing and characterisation of the clutch (epsE) and biomaterial synthesis (SB - signal sequence & biomaterial).

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3A.png"> <img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase3B.png"></html>

Phase 4

Combining of light induction and epsE/biomaterial expression, and testing of feasibility.

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4A.png"> <img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/Phase4B.png"></html>

Final Construct

Combination of light sensing and light-induced expression of epsE and biomaterial. Each gene has its own promoter because in B. subtilis it has been shown that levels of expression drop as one moves along an operon.

<html><img width="100%" src="http://i59.photobucket.com/albums/g305/Timpski/S1L.png"></html>

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