To build more complex cellular systems, new tools and techniques are required. We are generating standardized parts, tools, and techniques for the gram-positive chassis B. subtillis. Easy to handle and transform, this bacterium offers many adantages to E. coli', including the ability to secrete proteins and integrate DNA into the chromosome. We have designed, built, and submitted gram-positive RBSes, promoters, and shuttle vectors.As a part of this work we have confirmed single copy chromosomal insertion, demonstrated InFusion assembly, and characterized an improved GFP variant. You can find our Bacillus protocols here.

Improved GFP

Superfolder GFP along with other variants under UV light

We created a codon-optimized and biobricked version of the 'Superfolder' GFP engineered by Pédelacq et al (2006). This GFP variant folds faster and glows brighter than other GFP variants. Read more...

Creating new Biobrick-compatible B.subtilis vectors

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.

pBSint1 Vector pBSep1 Vector

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


B. subtilis Promoters Designed

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

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

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