UCSF

The ability to engineer enzymes has wide ranging applications in industry and biotechnology. Recently, methods and tools have been developed to engineer enzymes to catalyze a broad range of reactions and conditions. Despite these achievements, the task of enzyme engineering remains quite challenging due to the vast amount of possible sequences and interactions within a protein. This dissertation describes droplet microfluidic tools and methods for ultra-high-throughput screening of enzymes. By screening through variants faster and with less reagent, we increase our chances of finding improved variants. We first describe a fabrication method to make 3-D double emulsion devices in PDMS. We then describe a droplet microfluidic method for high throughput sequence-function mapping. Finally, we detail a detergent-free method to lyse cells in droplets using electroporation. By enabling us to screen enzymes at high throughput, these droplet microfluidic technologies can be a powerful tool for enzyme discovery.