Team:Johns Hopkins-Wetware/Project

Synthetic biology is helping to solve problems such as malnutrition and disease through the production of compounds such vitamins and medications. Using microorganisms to produce these compounds can lower their cost and make them more readily available. This year, the Johns Hopkins wetware team presents tools for better controlling cellular processes, which will allow optimization of the biosynthetic pathways manufacturing these compounds. Our two projects are an ethanol level self-regulation system, and a system for optogenetic control of protein function.

In industrial fermentation, the buildup of toxic intermediates and byproducts keeps productivity from reaching its full potential. In yeast, ethanol toxicity is the major chemical stress. To reduce ethanol stress, we constructed an ethanol-level self-regulation system consisting of the human cytochrome p450 CYP2E1 driven by a library of ethanol-induced promoters. CYP2E1 catalyzes the conversion of ethanol to acetaldehyde and then to acetate. When the ethanol level exceeds the optimal level, expression of CYP2E1 is triggered, which breaks down the excess ethanol.

The ability to quickly activate and deactivate proteins can be used to regulate flux through a biosynthetic pathway in order to minimize stress on the organism and possibly optimize yield. The use of light as a control mechanism has the advantages of being fast-acting, reversible, and amenable to automation in industrial applications. To demonstrate the ability to control proteins using light, we constructed a system for light-induced cell cycle arrest in yeast. We used the two-protein ePDZ/LOVpep light-induced dimerization system designed by Glotzer (citation) in two modules: One demonstrating light-induced activation of protein function by restoring a complete protein from two non-functional halves, and the other demonstrating light-induced deactivation of protein function by protein re-localization.