Directed evolution and solid phase enzyme screening

Edward J. Bylina; Christina L. Grek; William J. Coleman; Douglas C. Youvan

doi:10.1117/12.380511

22 March 2000 Directed evolution and solid phase enzyme screening

Edward J. Bylina, Christina L. Grek, William J. Coleman, Douglas C. Youvan

Proceedings Volume 3926, Advances in Nucleic Acid and Protein Analyses, Manipulation, and Sequencing; (2000) https://doi.org/10.1117/12.380511
Event: BiOS 2000 The International Symposium on Biomedical Optics, 2000, San Jose, CA, United States

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.

Citation Download Citation

Edward J. Bylina, Christina L. Grek, William J. Coleman, and Douglas C. Youvan "Directed evolution and solid phase enzyme screening", Proc. SPIE 3926, Advances in Nucleic Acid and Protein Analyses, Manipulation, and Sequencing, (22 March 2000); https://doi.org/10.1117/12.380511

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