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) Read more...

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

Read more...

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
  • Successfully expressed in an episomal vector...
• 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

Read more about the constructs made... Read more about the primers used...

Information about the B. subtilis vectors can be found [[Team:Cambridge/Signalling/Vectors here].