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

[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Cambridge Vectors submitted:] 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)

Observation with microscope (x20) : B.S. transformed with ECE166

Transformation with integration vectors

ECE153 and ECE112 : Transformation in IA751 : Amylase test

IA751 transformed with ECE153 : SUCCESFUL TRANSFORMATION, no zone of clearing

IA751 transformed with ECE112 : SUCCESFUL TRANSFORMATION for 4 colonies out of 5, no zone of clearing

B. subtilis Promoters Designed: [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Cambridge Biobricks submitted:]

• Pupp - a strong constitutive Bacillus cereus promoter present on B. subtilis vector ECE166
• Ppac - constitutive, present on B. subtilis vector ECE151
• Pspac - IPTG 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 [[Team:Cambridge/Signalling/Vectors here].

The Schematic:

1. PCR the 4 promoters out of the corresponding B.subtilis vectors using primers containing the standard biobrick restriction sites
2. Transform TOP10 with Biobrick I732007
3. Single colony PCR the transformed TOP10 cells and miniprep the colony containing I732007
4. Digest the promoter with EcoRI and SpeI
5. Digest I732007 plasmid with EcoRI and XbaI
6. Ligate the promoter to the I732007 backbone
7. Transform TOP10 cells with the new construct which should have Ampicillin resistance
8. Single colony PCR for successfully transformed cells and miniprep for more of new construct
9. Digest the new construct with EcoRI and BamHI
10. Digest ECE112 vector with EcoRI and BamHI
11. Dirty ligation of the promoter to ECE112 vector
12. Single colony PCR for the plasmid and miniprep
13. Transform B. subtilis with the plasmid which contains amyE for integration and lacZ for beta-galactosidase assay
14. Perform beta- galatosidase assay!