My overall goal is to gain insight into how evolutionary rewiring of genetic networks can generate phenotypic diversity. In order to study this, I propose to use synthetic, combinatorial-based, plasmid-bourne genetic networks in E. coli. Similar to computer logic gates, these networks will utilize two small-molecule inducers as inputs and a selectable marker as output. The networks' combinatorial architecture allows for a wide variety of logics. Through directed evolution, a combinatorial collection of networks will be pared down by selection in parallel assays to satisfy particular logics. The successful, optimized plasmid networks for each logic will be sequenced and analyzed. Then, starting with specific plasmid networks with corresponding logics, additional rounds of directed evolution will be undertaken to channel each in parallel into a completely different behavior. Sequencing and comparison with the initial plasmid will be performed to understand the underlying network changes and evolutionary path taken. In addition to addressing fundamental biological issues, evolutionary optimization of network logics should also provide a potentially powerful bioengineering tool.
