title: Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution.
abstract: We demonstrate the utility of a microfluidic platform in which water-in-oil droplet compartments serve to miniaturize cell lysate assays by a million-fold for directed enzyme evolution. Screening hydrolytic activities of a promiscuous sulfatase demonstrates that this extreme miniaturization to the single-cell level does not come at a high price in signal quality. Moreover, the quantitative readout delivers a level of precision previously limited to screening methodologies with restricted throughput. The sorting of 3 × 10(7) monodisperse droplets per round of evolution leads to the enrichment of clones with improvements in activity (6-fold) and expression (6-fold). The detection of subtle differences in a larger number of screened clones provides the combination of high sensitivity and high-throughput needed to rescue a stalled directed evolution experiment and make it viable.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
authors.forename: Balint
authors.initials: B
authors.lastname: Kintses
authors.forename: Christopher
authors.initials: C
authors.lastname: Hein
authors.forename: Mark F
authors.initials: MF
authors.lastname: Mohamed
authors.forename: Martin
authors.initials: M
authors.lastname: Fischlechner
authors.forename: Fabienne
authors.initials: F
authors.lastname: Courtois
authors.forename: Céline
authors.initials: C
authors.lastname: Lainé
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
mesh_terms: D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000378:metabolism; D019793:Fluorescein / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D008904:Miniaturization; D009821:Oils / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013429:Sulfatases / Q000235:genetics / Q000378:metabolism*; D014867:Water / Q000737:chemistry
chemical_list: D009821:Oils; D011994:Recombinant Proteins; D014867:Water; D013429:Sulfatases; D019793:Fluorescein