Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS).

ABSTRACT: Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD⁺-dependent amino acid dehydrogenases. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic r

SUBMITTER: Gielen F 

PROVIDER: S-EPMC5127370 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature