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New Bacterial Communication Lines by Directed Evolution of Lux R

Frances H. Arnold, Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, Cynthia Collins, Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th St, Troy, NY 12180, Cara Tracewell, Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, and Jared Leadbetter, Env. Microbiology, California Institute of Technology, Pasadena, CA 91125.

Members of the family of LuxR transcriptional activators involved in bacterial quorum sensing systems activate gene expression in response to binding an acyl homoserine lactone signal. Variants of LuxR that respond to new signals or activate transcription at new promoter sequences will be useful components for constructing cell-cell communication systems that program population-level behaviors in multicellular systems. We have developed a dual selection system to identify LuxR variants exhibiting a desired response, or lack thereof, to specific signals and promoter sequences. Specificity is required for establishing communication lines that do not crosstalk. Directed evolution with positive selection for response to a new signal or sequence usually generates broadened specificityŚrefocusing specificity requires negative selection to delete the original function. With dual selection, we have been able to engineer a series of LuxR signaling and DNA binding domains. These domains can be recombined to generate an even greater diversity of lines for cellular communications.