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Working with B.subtilis
Building on the work of the last year's Cambridge iGEM team, we are exploring applications of the gram-positive chassis B.subtillis. Easy to handle and transform, this bacterium is much better to use than E.coli wherever protein import/export is concerned, e.g. in our signalling project. An important part of our effort is to establish standard protocols and parts to work in B.subtillis, characterise control elements, and develop new vectors. You can find our Bacillus protocols here.
Creating new Biobrick-compatible B.subtilis vectors
We are using the In-Fusion™ PCR method from ClonTech to create new Biobrick-compatible integrative and episomal vectors. These vectors will allow us to insert Biobricks into the Bacillus subtilis genome at the AmyE locus and as a 3-5 copy plasmid that will replicate autonomously in Bacillus.
Successful transformation of Bacillus
Transformation with episomal vectors
ECE166 : High-level constitutive expression of a Green Fluorescent Protein (with the promoter PupP)
Transformation with integration vectors
ECE153 and ECE112 : Transformation in IA751 : Amylase test
Testing B.subtilis promoters and RBSs
We are seeking to test the expression strength and response to inducers in several promoters in B.subtilis, in combination with different Bacillus-specific ribosome binding sites.
B. subtilis Promoter Testing with Beta-galactosidase Assay
We are aiming to characterize four different Bacillus subtilis promoters on the ECE112 backbone using the beta-galactosidase assay. Protocol for beta-galactosidase assay as described here
B. subtilis Promoters to be Tested:
Information about the B. subtilis vectors can be found here
Links to Cambridge 2007 Wiki