(175f) Repressor-Based Tools for Cell-Cell Communication in Synthetic Biology

A grand challenge in synthetic biology is the development of novel intercellular signaling systems. We have engineered the acyl-homoserine lactone (AHL)-dependent transcriptional repressor, EsaR, and the esa promoter for use in synthetic microbial communities. The availability of intercellular communication-dependent repressors will enable new circuits and network behaviors not feasible with existing parts (e.g., the transcriptional activator LuxR). We have used directed evolution to identify a number of EsaR variants that respond to AHL concentrations between 5 and 10,000 nM. We have also engineered new EsaR dependent promoters. Here we have taken advantage of the AHL-dependent DNA-binding activity of EsaR to build synthetic AND gates that recognize both an extracellular signal and cell density. We have characterized our multi-input promoters and have found that activity can be tuned by varying AHL production, AHL sensitivity and addition of an endogenous inducer molecule.  These new transcriptional repressors and promoters will enable construction of novel engineered density-dependent or multicellular systems for metabolic engineering applications.