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Emulsion-based directed evolution of enzymes and proteins in yeast

Gardner, Elizabeth C. and Watkins, Ella J. and Gollihar, Jimmy and Ellington, Andrew D. (2020) Emulsion-based directed evolution of enzymes and proteins in yeast. In: Enzyme Engineering and Evolution: General Methods. Methods in Enzymology. No.643. Academic Press , Cambridge, MA, pp. 87-110. ISBN 978-0-12-821149-6.

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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.

Item Type:Book Section
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Watkins, Ella J.0000-0002-7365-5791
Additional Information:© 2020 Elsevier Inc. Available online 13 May 2020. This work was supported by NSF SemiSynBio Award 1807369. Special thanks to Dr. Katy Kao for transcribing the first draft of the protocol.
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Subject Keywords:Selection; Screening; Directed evolution; Emulsions; Emulsion droplets; Emulsion PCR; Protein engineering; Cellular compartmentalization
Series Name:Methods in Enzymology
Issue or Number:643
Record Number:CaltechAUTHORS:20200515-132802344
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Official Citation:Elizabeth C. Gardner, Ella J. Watkins, Jimmy Gollihar, Andrew D. Ellington, Chapter Four - Emulsion-based directed evolution of enzymes and proteins in yeast, Editor(s): Dan S. Tawfik, Methods in Enzymology, Academic Press, Volume 643, 2020, Pages 87-110, ISSN 0076-6879, ISBN 9780128211496, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103240
Deposited By: Tony Diaz
Deposited On:15 May 2020 20:57
Last Modified:16 Nov 2021 18:19

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