Cellular circuits in synthetic biology often depend on inducible regulators like the lac and tet repressors. However, increased circuit complexity is limited by the availability of orthogonal regulators. We present a method for generation of directed libraries of regulators for binding of DNA and RNA sequences in E. coli. Our unique logic-based selection 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). In addition, we have isolated point mutants that increase binding of repressors to RNA aptamers in the 5'-untranslated region of mRNA. We leverage the technologies of multiplex oligo synthesis, recombineering, and next-generation sequencing to create libraries and glean sequence-activity relationship data from selections to iterate evolution in a rational, directed way.

Engineering transcriptional and post-transcriptional repression for synthetic cellular circuits

249th National Meeting (2015)

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