Microfluidic Compartmentalized Directed Evolution

SummaryDirected evolution studies often make use of water-in-oil compartments, which conventionally are prepared by bulk emulsification, a crude process that generates nonuniform droplets and can damage biochemical reagents. A microfluidic emulsification circuit was devised that generates uniform water-in-oil droplets (21.9 ± 0.8 μm radius) with high throughput (107–108 droplets per hour). The circuit contains a radial array of aqueous flow nozzles that intersect a surrounding oil flow channel. This device was used to evolve RNA enzymes with RNA ligase activity, selecting enzymes that could resist inhibition by neomycin. Each molecule in the population had the opportunity to undergo 108-fold selective amplification within its respective compartment. Then the progeny RNAs were harvested and used to seed new compartments. During five rounds of this procedure, the enzymes acquired mutations that conferred resistance to neomycin and caused some enzymes to become dependent on neomycin for optimal activity.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (326 K)Download as PowerPoint slideHighlights► A microfluidic device was developed that generates highly uniform water-in-oil emulsions for use in directed evolution experiments ► The device generates tens of millions of droplets per hour that vary in diameter by less than 4% ► The device was used to evolve RNA enzymes with RNA ligase activity, selecting enzymes that could resist inhibition by neomycin ► The evolved enzymes acquired resistance to neomycin and some became dependent on neomycin for optimal activity