1. Bacillus subtilis expression/secretion systems
iGEM meeting

2. Current status of B. subtilis as an iGEM chassis
Under development by a couple of iGEM teams

3. Current status of B. subtilis as an iGEM chassis
Rationale (direct quote from Cambridge team wesite)
Since they do not have a second membrane, they will secrete and absorb substances more efficiently than Gram-negative bacteria.
B. subtilis can be easily transformed by the natural competency method.
B. subtilis is more motile than E. coli so will be more suitable for experiments on cell movement.
Since B. subtilis be easily cultured along the lines of standard E. coli protocols, not much new equipment will be required.
B. subtilis is a Class I contaminant (US EPA webpage), so it can be used without potential health or environmental risks.

4. Details of Cambridge subtilis project
Modifying existing B. subtilis integrational and shuttle plasmids to have the required BioBrick restriction sites, so that BioBricks can be first assembled in a suitable E. coli chassis and then transformed into B. subtilis
Investigating experimental protocols for dealing with B. subtilis
Characterizing the strength of present E. coli promoters in B. subtilis
Creating BioBricks of promoters which work very well in both E. coli and B. subtilis
Contributing new integrational vectors that will allow the insertion of novel genes into the B. subtilis host
, direct quote

5. Problems faced by Cambridge team
E. coli and B. subtilis origins and promoters are not generally interchangeable.
They’re working on “bio-bricking” promoters that can work in both (these definitely exist!)
There are “shuttle vectors” that can replicate in both (and there have been for >2 decades!)
All this and more (including optimized transformation protocol) included on their website

6. B. subtilis secretion pathways
Harwood and Cranenberg, 2008

7. Molecular requirements for protein secretion in B. subtilis
Signal peptide at N-terminus of secreted protein – average ~30 aa

8. B. subtilis expression-secretion system
Strain WB800 – deletion of 8 extracellular proteases
Strain WB800HM[pEPP] – WB800 + overexpression of intracellular and extracellular chaperones– can improve expression levels (Wu et al, 2002)

9. B. subtilis expression/secretion vectors: an example
Shuttle vector for Ec and Bs.
Nice MCS
Contains strong secretion sequence
Strong constitutive promoters
(Brockmeier et al (2006))

10. Inducible B. subtilis expression vectors
ITPG-inducible
(MoBiTech)
xylose-inducible

11. B. subtilis integration vectors

12. Availability of strains and vectors
Strains: 897 B. subtilis strains, but not WB800  Would probably have to get it from another researcher. UC Davis, U. Calgary, Germany, Netherlands, China, Japan)
Vectors: Chromosomal integration vectors available from BGSC. Others—may need to contact individual researchers