Ultrahigh-Throughput Screening of Single-Cell Lysates for Directed Evolution and Functional Metagenomics

Abstract

The success of ultrahigh-throughput screening experiments in directed evolution or functional metagenomics strongly depends on the availability of efficient technologies for the quantitative testing of a large number of variants. With advanced robotics, libraries of up to 10⁵ clones can be screened per day as colonies on agar plates or cell lysates in microwell plates, albeit at high cost of capital, manpower and consumables. These cost considerations and the general need for high-throughput make miniaturization of assay volumes attractive. To provide a general solution to maintain genotype-phenotype linkage, biochemical assays have been compartmentalized into water-in-oil droplets. This chapter presents a microfluidic workflow that translates a frequently used screening procedure consisting of cytoplasmic/periplasmic protein expression and cell lysis to the single cell level in water-in-oil droplet compartments. These droplets are sorted based on reaction progress by fluorescence measurements at the picoliter scale.

Keywords: Assay miniaturization; Directed evolution; Functional metagenomics; Hydrolase; In vitro compartmentalization; Microfluidic droplets; Microfluidics; Single-cell; Ultrahigh-throughput screening.