I will describe our efforts to construct synthetic multicellular systems in which bacterial populations communicate with each other and their environment via engineered signaling pathways. One example is a ‘microbial consensus consortium' (MCC) whose members converse bi-directionally by exchanging acyl-homoserine lactone (acyl-HSL) signals. The consortium generates a gene expression response if and only if both populations are present at sufficient cell densities (consensus); the overall response cannot be generated by either population alone (consortium). The MCC functions in diverse growth modes, including in a biofilm where it sustains its MCC response for several days. Other examples include predator-prey and a pattern-forming systems. These synthetic multicellular systems use signaling components (signal synthesizers and receivers) from bacterial quorum sensing systems. Members of the LuxR family of transcriptional activators activate gene expression in response to binding acyl-HSL signals. We are creating new components in the laboratory by directed evolution of these natural elements. LuxR analogs that respond to new signals or activate transcription at new promoter sequences are needed for constructing the next generation of cell-cell communication systems. 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 specificity—refocusing specificity requires negative selection to delete the original function. We have developed a dual selection system to identify LuxR variants exhibiting a desired response, or lack thereof, to specific signals and promoter sequences. 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. This work is a collaboration with the Ron Weiss laboratory at Princeton University.

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