Project Summary: The ability to use light to control biological processes has enabled the careful dissection of biology and could be developed into future treatments for blindness, cancer, and many other diseases. Systems that respond to red to near-infrared (NIR) light are particularly useful because those wavelengths efficiently penetrate tissue, but these systems can be greatly improved by engineering them to efficiently bind endogenous chromophores or to be more sensitive to light. By using a new continuous evolution system, OrthoRep, we will evolve the Phytochrome B (PhyB) optogenetic tool to use biliverdin (which is present in every cell) as a chromophore and evolve versions that are activated by different wavelengths of NIR light. This will be accomplished by modifying the Automated Continuous Evolution (ACE) that can automatically apply evolutionary pressure as the gene of the optogenetic tool continuously mutates. Because PhyB will respond to light by activating a gene essential for survival, it will be possible to evolve the activation wavelength, the amount of light that is required to activate the gene, and the level of the activation once illuminated. Evolved PhyB mutants that have useful properties will be validated/characterized biochemically and for controlling genes in mammalian cells. The resulting optogenetic tools will enable new opportunities for in vivo optogenetics and the modified ACE can be used to develop many other types of optogenetic tools, optical sensors, and more.