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Proceedings Paper

Directed evolution and solid phase enzyme screening
Author(s): Edward J. Bylina; Christina L. Grek; William J. Coleman; Douglas C. Youvan
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Paper Abstract

A new digital imaging spectrophotometer and a series of colorimetric solid phase arrays have been developed to screen bacterial libraries expressing mutagenized enzymes undergoing directed evolution. This high-throughput solid- phase array system (known as `Kcat Technology') can detect less than a 20% difference in enzyme rates within microcolonies grown at a nearly confluent density of 500 colonies per cm2 on an assay disk. Each microcolony is analyzed simultaneously at single-pixel resolution (1.5 megapixels; 75 micron/pixel), requiring less than 100 nanoliters of substrate per measurement, a 1000-fold reduction over conventional liquid phase assays. Here we report the successful identification of variants of Agrobacterium (beta) -glucosidase--a glycosidase with broad substrate specificity that favors cleavage of glucosides over galactosides--by simultaneously assaying two different substrates tagged with spectrally distinct chromogenic reporters.

Paper Details

Date Published: 22 March 2000
PDF: 6 pages
Proc. SPIE 3926, Advances in Nucleic Acid and Protein Analyses, Manipulation, and Sequencing, (22 March 2000); doi: 10.1117/12.380511
Show Author Affiliations
Edward J. Bylina, KAIROS Scientific, Inc. (United States)
Christina L. Grek, KAIROS Scientific, Inc. (United States)
William J. Coleman, KAIROS Scientific, Inc. (United States)
Douglas C. Youvan, KAIROS Scientific, Inc. (United States)

Published in SPIE Proceedings Vol. 3926:
Advances in Nucleic Acid and Protein Analyses, Manipulation, and Sequencing
Patrick A. Limbach; John C. Owicki; Ramesh Raghavachari; Weihong Tan, Editor(s)

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