Team:Open Science Global/Wetware/Our-Strains
Our Strains
Bacillus subtilis and Pichia pastoris are our platform strains, because scalable protein extraction from E. coli requires expensive equipment.
Escherichia coli, the most widely used workhorse for molecular and synthetic biology, does not secrete proteins efficiently. As a result, large-scale recombinant protein production and purification in E. coli generally requires equipment for cell lysis (a sonicator or french press) and separation of soluble proteins from the fragments of lysed cells (a refrigerated centrifuge). This equipment can cost thousands to tens of thousands of dollars even for laboratory-scale preparations.
We therefore chose Bacillus subtilis and Pichia pastoris (Komagataella pastoris) as the platform organisms for our wetware designs. B. subtilis is a gram-positive bacterium, and P. pastoris is a yeast. Both B. subtilis and P. pastoris are Generally Regarded As Safe by the United States Food and Drug Administration. Both are capable of high capacity (grams per liter) secretion of recombinant proteins, and are widely used in the biotechnology industry for recombinant protein production.
Of the two, we focused most of our design efforts on B. subtilis, for three reasons. First, B. subtilis grows more quickly, which should make design-build-test cycles faster. Second, it is important that we be able to freely share any cell lines we engineer to produce useful proteins. While we have been able to purchase Dan Ziegler’s patent-free and Material Transfer Agreement (MTA)-free B. subtilis protein expression strains from the Bacillus Genetic Stock Center (BGSC), we have not yet been able to obtain IP-free Pichia expression strains, either under an OpenMTA or without an MTA. Finally, our team’s primary PI Scott Pownall (with contributions from team co-lead Isaac Larkin) already designed for FreeGenes an open, IP-free wetware collection for bioengineering Saccharomyces cerevisae and Pichia pastoris: the Open Yeast Collection (OYC). Since open wetware tools for engineering yeast already exist, we decided it would be most useful for our wetware design efforts to focus on building a complementary Bacillus toolkit, taking design inspiration from OYC.