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High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions.
Mastrobattista, Enrico; Taly, Valerie; Chanudet, Estelle; Treacy, Patrick; Kelly, Bernard T; Griffiths, Andrew D.
Afiliação
  • Mastrobattista E; Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
Chem Biol ; 12(12): 1291-300, 2005 Dec.
Article em En | MEDLINE | ID: mdl-16356846
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.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Biblioteca Gênica / Beta-Galactosidase / Evolução Molecular Direcionada / Emulsões / Escherichia coli / Citometria de Fluxo Tipo de estudo: Diagnostic_studies / Prognostic_studies / Screening_studies Idioma: En Ano de publicação: 2005 Tipo de documento: Article
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Biblioteca Gênica / Beta-Galactosidase / Evolução Molecular Direcionada / Emulsões / Escherichia coli / Citometria de Fluxo Tipo de estudo: Diagnostic_studies / Prognostic_studies / Screening_studies Idioma: En Ano de publicação: 2005 Tipo de documento: Article