Bacillus subtilis expressionsecretion systems 6 1 08 i

Bacillus subtilis expression/secretion systems 6 -1 -08 i. GEM meeting

Current status of B. subtilis as an i. GEM chassis • Under development by a couple of i. GEM teams

Current status of B. subtilis as an i. GEM 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. http: //www. ccbi. cam. ac. uk/i. GEM 2007/index. php/Bacillus_subtilis_S yn. Bio_chassis

Details of Cambridge subtilis project • Modifying existing B. subtilis integrational and shuttle plasmids to have the required Bio. Brick restriction sites, so that Bio. Bricks 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 Bio. Bricks 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 http: //www. ccbi. cam. ac. uk/i. GEM 2007/index. php/Bacillus_s ubtilis_Syn. Bio_chassis , direct quote

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

B. subtilis secretion pathways Harwood and Cranenberg, 2008

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

B. subtilis expression-secretion system • Strain WB 800 – deletion of 8 extracellular proteases • Strain WB 800 HM[p. EPP] – WB 800 + overexpression of intracellular and extracellular chaperones– can improve expression levels (Wu et al, 2002)

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))

Inducible B. subtilis expression vectors ITPG-inducible (Mo. Bi. Tech) xylose-inducible

B. subtilis integration vectors

Availability of strains and vectors • Strains: 897 B. subtilis strains, but not WB 800 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