S146: Directed evolution of quorum-sensing repressors for programmed intercellular communication in synthetic microbial consortia

One of the key challenges in the field of synthetic biology is to engineer not only single cells but communities that exhibit coordinated, population-level behaviors. Synthetic biologists have employed components from acyl-homoserine lactone (AHL)-based quorum-sensing (QS) systems in their endeavors to build multicellular systems. To date, the regulators available in the toolbox of QS regulatory parts for engineering intercellular communication have been limited to transcriptional activators, most frequently the AHL-dependent activator LuxR. Transcriptional activators function by recruiting RNA polymerase (RNAp) to a promoter sequence and thus require specific interactions with RNAp. Transcriptional repressors bind to operator regions within a promoter and inhibit the initiation of transcription. To expand our toolbox of regulatory parts for cell-cell communiaction, we have engineered the AHL-dependent transcriptional repressor, EsaR. Wild-type EsaR requires micromolar concentrations of AHL to achieve complete derepression, while the LuxR will activate gene expression in the presence of nanomolar concentrations of AHL. Therefore, we built libraries of esaR mutants and identified EsaR variants with increased AHL sensitivity using an ON/OFF screening system. We first screened for an absence of gene expression to identify EsaR variants that retained the ability to repress gene expression (OFF screening), and then screened for reporter gene expression in the presence of nanomolar concentrations of AHL (ON screening). We have identified EsaR variants that respond to a range of AHL concentrations, from 10 nM to 1 uM. These new parts are enabling work exploring the roles of regulatory mechanisms and network architectures for intercellular communication in multicellular systems.