Microfluidic Compartmentalized Directed Evolution

Overview

Journal Chem Biol

Publisher Elsevier

Specialties Biochemistry
Biology
Chemistry

Date 2010 Jul 28

PMID 20659684

Citations 26

Authors

Brian M Paegel

Gerald F Joyce

Affiliations

Soon will be listed here.

Abstract

Directed 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 microm radius) with high throughput (10(7)-10(8) 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 10(8)-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.

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