PROJECT SUMMARY / ABSTRACT The goal of this project is to demonstrate the feasibility of a novel technology that is expected to boost yields from pharmaceutical industrial insulin production by greater than four-fold over conventional methods. Higher yields will lower production costs, generating savings that will ultimately be passed onto the millions of diabetic patients who require daily insulin injections. The technology is called MiST (Microbial Stem Cell Technology), and is proprietary to AsmicA, the small business interest in this STTR phase I project proposal, in collaboration with scientists at the University of Wyoming. In MiST cell cultures, a bioreactor vessel is populated with a small but persistent population of bacterial “stem cells”, which always divide asymmetrically into one stem cell and another that differentiates into healthy new insulin-producing “factory cells”. Owing to continual generation of robust, rapidly dividing, and highly productive new factory cells, MiST cultures produce a higher total number of factory cells than conventional cultures, leading to substantially higher final yield. The rapidly dividing populations of stem cells and young factory cells in MiST cultures also compete against the proliferation of non-productive “cheater mutants” that ultimately spoil production, thereby increasing yields by extending the time of product synthesis. The aims of this project are to express insulin in MiST strains (Aim #1) and determine whether these strains generate the expected enhancement in yield in a laboratory scale bioreactor (Aim #2). This feasibility study will serve as the basis for entering into contractual partnerships with commercial insulin manufacturers, including up-and-coming generic companies. Here, the goal will be to generate industrial MiST strains and adapt MiST culturing methods for high-yield industrial scale production.