Emulsion-based directed evolution of enzymes and proteins in yeast

Elizabeth C. Gardner, Ella J. Watkins, Jimmy Gollihar, Andrew D. Ellington

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Emulsion-based selections are a unique type of directed evolution method that overcome common bottlenecks associated with purely in vivo selections. For example, emulsions including cell-free translation machinery can be useful for expression of toxic genes. However, not all cell types can efficiently produce protein in vitro, for example, the eukaryotic microbe Saccharomyces cerevisiae. compartmentalized self replication (CSR) and compartmentalized partnered replication (CPR) are two emulsion-based selection schemes that leverage the advantages of both in vivo and in vitro selections by compartmentalizing cells in water-in-oil droplets. Previous implementations of these methods utilized bacterial hosts, which has limited the technology to the directed evolution of proteins that can be heterologously expressed in prokaryotic systems. To expand the repertoire of targets that can be evolved, we have adapted emulsion-based PCR selections to be compatible with a eukaryotic host.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsDan S. Tawfik
PublisherAcademic Press
Pages87-110
Number of pages24
ISBN (Print)9780128211496
DOIs
StatePublished - 2020

Publication series

NameMethods in Enzymology
Volume643
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Cellular compartmentalization
  • Directed evolution
  • Emulsion PCR
  • Emulsion droplets
  • Emulsions
  • Protein engineering
  • Screening
  • Selection

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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