In vitro compartmentalization by double emulsions: Sorting and gene enrichment by fluorescence activated cell sorting

Kalia Bernath, Mingtan Hai, Enrico Mastrobattista, Andrew D. Griffiths, Shlomo Magdassi, Dan S. Tawfik*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

148 Scopus citations

Abstract

Water-in-oil (w/o) emulsions can be used to compartmentalize and select large gene libraries for a predetermined function. The aqueous droplets of the w/o emulsion function as cell-like compartments in each of which a single gene is transcribed and translated to give multiple copies of the protein (e.g., an enzyme) it encodes. While compartmentalization ensures that the gene, the protein it encodes, and the products of the activity of this protein remain linked, it does not directly afford a way of selecting for the desired activity. Here we show that re-emulsification of w/o emulsions gives water-in-oil-in- water (w/o/w) emulsions with an external (continuous) water phase through which droplets containing fluorescent markers can be isolated by fluorescence- activated cell sorting (FACS). These w/o/w emulsions can be sorted by FACS, while the content of the aqueous droplets of the primary w/o emulsion remains intact. Consequently, genes embedded in these water droplets together with a fluorescent marker can be isolated and enriched from an excess of genes embedded in water droplets without a fluorescent marker. The ability of FACS instruments to sort up to 40,000 events per second may endow this technology a wide potential in the area of high-throughput screening and the directed evolution of enzymes.

Original languageAmerican English
Pages (from-to)151-157
Number of pages7
JournalAnalytical Biochemistry
Volume325
Issue number1
DOIs
StatePublished - 1 Feb 2004

Bibliographical note

Funding Information:
Funding by an Israeli Ministry of Science (IMOS) grant to D.S.T. and S.M. is gratefully acknowledged. A.D.G. and D.S.T. are members of the European Network on Directed Evolution of Functional Proteins (ENDIRPRO).

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