title: High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions.
abstract: We describe a completely in vitro high-throughput screening system for directed evolution of enzymes based on in vitro compartmentalization (IVC). Single genes are transcribed and translated inside the aqueous droplets of a water-in-oil emulsion. Enzyme activity generates a fluorescent product and, after conversion into a water-in-oil-in-water double emulsion, fluorescent droplets are sorted using a fluorescence-activated cell sorter (FACS). Earlier in vivo studies have demonstrated that Ebg, a protein of unknown function, can evolve to allow Escherichia coli lacking the lacZ beta-galactosidase gene to grow on lactose. Here we demonstrate that we can evolve Ebg into an enzyme with significant beta-galactosidase activity in vitro. Only two specific mutations were ever seen to provide this improvement in Ebg beta-galactosidase activity in vivo. In contrast, nearly all the improved beta-galactosidases selected in vitro resulted from different mutations.
authors.affiliation: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
authors.forename: Enrico
authors.initials: E
authors.lastname: Mastrobattista
authors.forename: Valerie
authors.initials: V
authors.lastname: Taly
authors.forename: Estelle
authors.initials: E
authors.lastname: Chanudet
authors.forename: Patrick
authors.initials: P
authors.lastname: Treacy
authors.forename: Bernard T
authors.initials: BT
authors.lastname: Kelly
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Griffiths
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods*; D004655:Emulsions; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D015723:Gene Library; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering / Q000379:methods; D012097:Repressor Proteins / Q000235:genetics; D013997:Time Factors; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: C062847:EbgR protein, E coli; D004655:Emulsions; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D001616:beta-Galactosidase