Positive feedback is a common gene regulatory motif that is responsible for gene-expression bistability in both prokaryotes and eukaryotes. Artificial positive feedback loops have also been used as genetic amplifiers for enhancing the responses of weak promoters, and in the creation of eukaryotic gene switches. In this presentation we will detail the construction and directed evolution of two positive feedback loops based on the LuxR transcriptional activator and its cognate promoter, PluxI. The wild-type positive feedback loops are completely activated at concentrations as low as 10 nM of 3-oxo-hexanoyl-homoserine lactone (OHHL). Directed evolution of the LuxR transcriptional activator increased the sensitivity of the feedback loops resulting in systems that are completely activated at OHHL concentrations as low as 5 nM, or approximately 3 molecules per cell. The responses of the positive feedback loops can also be modulated by adjusting inducer concentrations. We expect that these highly sensitive yet regulatable positive feedback loops will find various applications in industrial fermentation and gene therapy.

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