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.

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 using the beta-galactosidase assay. (Protocol for beta-galactosidase assay as described here

B. subtilis Promoters to be Tested:

• Pupp - constitutive, a Bacillus cereus promoter present on B. subtilis vector ECE166
• Ppac - constitutive, present on B. subtilis vector ECE151
• Pspac - lactose inducible, present on B. subtilis vector ECE151
• Pxyl - xylose inducible, present on B. subtilis vector ECE153

Information about the B. subtilis vectors can be found [here]

Links

• Chris French's protocol on transforming B. subtilis
• We have last year's protocol; not sure if it's trustworthy, need to compare with online resources
• Protocol from BCSG catalog
  • used in Cornell bacteriology course, probably should start here

B. Subtilis Transformation Protcol - Electroporation

Bongers et al. … of a Subtilin-Regulated Expression System in Bacillus subtilis: Strict Control of Gene Expression …. Applied and Environmental Microbiology (2005)

• SURE: handy expression system for B. subtilis
• What about feedback/regulation effects of subtilin on the cell

Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

• (Some strains of) Bacillus subtilis produces a gene called aiiA that degrades AHL molecules from gram-negative bacteria
• this is biobricked

Links to Cambridge 2007 Wiki

• B. subtilis group
• B. subtilis group: more info