Title of article :
High-Throughput Screening of Enzyme Libraries: Thiolactonases Evolved by Fluorescence-Activated Sorting of Single Cells in Emulsion Compartments Original Research Article
Author/Authors :
Amir Aharoni، نويسنده , , Gil Amitai، نويسنده , , Kalia Bernath، نويسنده , , Shlomo Magdassi، نويسنده , , Dan S. Tawfik، نويسنده ,
Issue Information :
ماهنامه با شماره پیاپی سال 2005
Pages :
9
From page :
1281
To page :
1289
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 >107 serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >104 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.
Journal title :
Chemistry and Biology
Serial Year :
2005
Journal title :
Chemistry and Biology
Record number :
1159127
Link To Document :
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