Phase 1: Testing of Constitutive Promoters
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.
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.
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.
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).
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.