Team:Imperial College/Cloning Strategy
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We will test 4 combinations of 2 constitutive promoters and 2 RBSs to characterise them. An antibiotic resistance cassette is placed at the 5' end of the construct, to prevent any readthrough by the native trancriptases from reaching the regulated BioBricks. | We will test 4 combinations of 2 constitutive promoters and 2 RBSs to characterise them. An antibiotic resistance cassette is placed at the 5' end of the construct, to prevent any readthrough by the native trancriptases from reaching the regulated BioBricks. | ||
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====== Phase 2: Testing of Inducible Promoters ====== | ====== Phase 2: Testing of Inducible Promoters ====== | ||
Promoters marked with a 'C' are chemically-inducible and those marked with an 'L' are light-inducible. Since YtvA responds to blue light, fluorescence from GFP may cause positive feedback. We will therefore be using RFP as a quantifiable output to test our light-inducible promoters instead. 'Rep' genes encode a repressor for the chemically-inducible promoters to stop leaky expression. | Promoters marked with a 'C' are chemically-inducible and those marked with an 'L' are light-inducible. Since YtvA responds to blue light, fluorescence from GFP may cause positive feedback. We will therefore be using RFP as a quantifiable output to test our light-inducible promoters instead. 'Rep' genes encode a repressor for the chemically-inducible promoters to stop leaky expression. | ||
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====== Phase 3: Clutch and Biomaterial Characterisation ====== | ====== Phase 3: Clutch and Biomaterial Characterisation ====== | ||
Testing and characterisation of the clutch (EpsE, produced by the ''epsE'' gene) and biomaterial synthesis (SB - signal peptide and biomaterial) using a chemically-inducible promoter. The promoter is otherwise constitutively repressed by Rep. | Testing and characterisation of the clutch (EpsE, produced by the ''epsE'' gene) and biomaterial synthesis (SB - signal peptide and biomaterial) using a chemically-inducible promoter. The promoter is otherwise constitutively repressed by Rep. | ||
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====== Phase 4: Device Characterisation ====== | ====== Phase 4: Device Characterisation ====== | ||
Combination of light induction and EpsE/biomaterial expression. The clutch (EpsE) and biomaterial are now under control of the light-inducible activator sigma B (via YtvA). | Combination of light induction and EpsE/biomaterial expression. The clutch (EpsE) and biomaterial are now under control of the light-inducible activator sigma B (via YtvA). | ||
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====== Final Construct: System Characterisation ====== | ====== Final Construct: System Characterisation ====== | ||
Combination of light sensing and light-induced expression of epsE and biomaterial. Each gene has its own promoter/RBS pair because in ''B. subtilis'', it has been shown that levels of expression decrease as one moves along an operon. | Combination of light sensing and light-induced expression of epsE and biomaterial. Each gene has its own promoter/RBS pair because in ''B. subtilis'', it has been shown that levels of expression decrease as one moves along an operon. | ||
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{{Imperial/EndPage|Wet_Lab|Protocols}} | {{Imperial/EndPage|Wet_Lab|Protocols}} |
Revision as of 16:01, 17 October 2008
Cloning Strategy
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