Project Summary/Abstract The unifying theme of our research on synthetic biology is to utilize the principles of Darwinian evolution to help us understand or expand the fundamental processes of life. As such, the application of directed evolution to alter, reconstitute, or expand these processes is paramount. Establishing an automated continuous evolution system comprised of continuous hypermutation of a gene of interest by a mutagenic T7 replisome, in combination with continuous culturing of its microbially tractable host E. coli, will greatly accelerate such evolution campaigns in our lab. Specifically, this will support our research on orthogonal tRNA/RS pairs for genetic code expansion, as well as the evolution of therapeutic proteases and enzymes.