IGEM:IMPERIAL/2008/New/Cloning Strategy

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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 to date. While the B. subtilis chassis offers us many advantages, working from the ground up presents many challenges.

Our cloning strategy is highly complex. In order to build increasingly complicated constructs for our final product, we need to build, test and characterise all the parts and devices leading to the final systems. The diagram below shows the critical pathway for our cloning strategy with a huge number of closely-linked steps.

A summary of the aims of each phase and constructs to be produced within each phase, is given 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.

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 as a quantifiable output as ytvA responds to blue light - GFP may cause positive feedback. 'Rep' genes encode a repressor for the chemically-inducible promoters to stop leaky expression.

Phase 3

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

Phase 4

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

Final Construct

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

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