TY - JOUR
T1 - High-throughput screening of enzyme libraries
T2 - Thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments
AU - Aharoni, Amir
AU - Amitai, Gil
AU - Bernath, Kalia
AU - Magdassi, Shlomo
AU - Tawfik, Dan S.
N1 - Funding Information:
We are very grateful to Eitan Ariel and Ayala Sharp for their devoted assistance with the FACS. Financial support from the Israel Science Foundation (ISF) through its Bikura grant, and the Israeli Ministry of Science (IMOS), are gratefully acknowledged. D.T. is the incumbent of the Elaine Blond Career Development Chair.
PY - 2005/12/1
Y1 - 2005/12/1
N2 - 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.
AB - 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.
UR - https://www.scopus.com/pages/publications/28844466075
U2 - 10.1016/j.chembiol.2005.09.012
DO - 10.1016/j.chembiol.2005.09.012
M3 - Article
AN - SCOPUS:28844466075
SN - 1074-5521
VL - 12
SP - 1281
EP - 1289
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 12
ER -