Directed evolution by in vitro compartmentalization

  • Miller O
  • Bernath K
  • Agresti J
 et al. 
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Abstract

The goal of in vitro compartmentalization (IVC) is to divide a large reaction between many microscopic compartments. This technique was first developed to generate "artificial cells" for the directed evolution of proteins. Typically, an aq. soln. of genes and an in vitro transcription-translation system is stirred (or homogenized) into an oil-surfactant mixt. to create a water-in-oil (w/o) emulsion with -1010 aq. droplets per mol of emulsion. The majority of droplets contain no more that a single gene along with all of the mol. machinery needed to express that gene. The expressed proteins and the products of their catalytic activities cannot leave the droplets, and so genotype is coupled to phenotype in vitro, making it possible to select very large libraries of genes (108-1011 genes). We describe the advantages and applications of IVC. A protocol for performing a directed evolution expt. by IVC makes use of one or more w/o emulsions. This procedure involves the generation of a gene library, the performance of a selection, and the subsequent recovery of the selected genes by PCR. We also describe two procedures for converting w/o emulsions to water-in-oil-in-water (w/o/w) emulsions for high-throughput screening using a fluorescence-activated cell sorter (FACS). Finally, we describe two methods for delivering substrates, regulators and other compds. to the preformed aq. droplets of w/o emulsion. on SciFinder (R)

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Authors

  • Gil AmitaiWeizmann Institute of Science

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  • Oliver J. Miller

  • Kalia Bernath

  • Jeremy J. Agresti

  • Bernard T. Kelly

  • Enrico Mastrobattista

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