(378f) Engineering Orthogonal Protein-Nucleic Acid Pairs for Cellular Circuits

Cellular circuits in synthetic biology often depend on inducible regulators like the lac and tet repressors. However, increased circuit complexity is currently limited by the availability of orthogonal regulators.  We present a method for generation of directed libraries of regulators for orthogonal binding of DNA and RNA sequences in E. coli.  Our unique selection-counterselection strategy allows for selection of repressors that do bind a target (i.e., novel) sequence but do not bind a pre-determined sequence of interest (e.g., wild type). We leverage the technologies of multiplex oligo synthesis and next-generation high-throughput sequencing to create libraries and glean sequence-activity relationship data from selections to iterate evolution in a rational, directed way.