High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions

Chem Biol. 2005 Dec;12(12):1291-300. doi: 10.1016/j.chembiol.2005.09.016.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Directed Molecular Evolution / methods*
  • Emulsions*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / genetics
  • Flow Cytometry / methods*
  • Gene Library*
  • Models, Molecular
  • Mutation
  • Protein Engineering / methods
  • Repressor Proteins / genetics
  • Time Factors
  • beta-Galactosidase / genetics*
  • beta-Galactosidase / metabolism


  • EbgR protein, E coli
  • Emulsions
  • Escherichia coli Proteins
  • Repressor Proteins
  • beta-Galactosidase