Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at ∼2-3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.
Keywords: DrOPS; droplet microfluidics; droplet sorting; enzyme engineering; high throughput screening.