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Chem Biol. 2005 Dec;12(12):1281-9.

High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments.

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1
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

Abstract

Single bacterial cells, each expressing a different library variant, were compartmentalized in aqueous droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzyme-coding genes. We demonstrate the directed evolution of new enzyme variants by screening >10(7) serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >10(4) enzyme molecules, in a volume of <10 femtoliter (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.

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PMID:
16356845
DOI:
10.1016/j.chembiol.2005.09.012
[Indexed for MEDLINE]
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