Directed evolution of custom polymerases using droplet microfluidics.

DNA polymerases are critical tools for a large number of emerging applications in biotechnology, but oftentimes polymerases with desired functions are not readily available. Directed evolution provides a possible solution to this problem by enabling the creation of engineered polymerases that are better equipped to recognize a given unnatural substrate. Here we report a microfluidic-based method for evolving new polymerase functions that involves ultrahigh throughput sorting of fluorescent water-in-oil (w/o) microdroplets. The workflow entails the expression of a diverse population of polymerase variants in E. coli, production of microfluidic droplets containing one or less E. coli, bacteria lysis to release the polymerase and encoding plasmid into the surrounding droplet, a fluorescence-based activity assay to identify variants with a desired activity, isolation of fluorescent droplets using a fluorescence activated droplet sorting (FADS) device, and plasmid recovery with DNA sequencing to determine the identity of the functional variants. This technique is amenable to any type of unnatural nucleic acid and/or polymerase function, including DNA-templated synthesis, reverse transcription, and replication.