Directed Evolution of an Array of Orthogonal Biosensors 

Small molecule-inducible transcription factors, or sensors, enable temporal control of toxic genes, provide the inputs for in vivo computation, and allow a host to sense and respond to its environment. In this ongoing work we endeavor to improve the characteristics of several sensors using a mixture of rational engineering and directed evolution. We have improved the dynamic range, sensitivity, and/or specificity of 18 sensors. As an example of the utility of these sensors, we have used five optimized sensors to independently control the expression of five components of a metabolic pathway. By varying the amount of each inducer, we can easily and rapidly optimize the expression profile of the pathway without the need to construct large libraries. To facilitate the adoption of multiplex, sensor-based genetic control, we have created a stable cell-line capable of responding to 11 inputs, thus enabling the modular control of multiple genetic outputs with minimal optimization.