Solid-Phase Screening Using Digital Image Analysis

Abstract

It is often possible to screen libraries of variant enzymes directly on agar plates. Colony-based solid-phase screening is an attractive option because of its relative ease and high throughput compared to liquid-phase screening in multi-well plates. As with any high throughput screening approach, a suitable colorimetric or fluorimetric assay must exist (or be developed) for the enzyme function in question. There are two major requirements that must be met for solid-phase screening to be feasible. First, the substrate must be able to be supplied as part of the growth medium, as a vapor, or within the cell itself. Second, the assay must be sufficiently sensitive that colonies expressing enzyme variants successfully evolved for the desired function will exhibit differences in color or fluorescence distinguishable from colonies expressing the wildtype/parental enzyme or unsuccessful mutants. Solid-phase assays have been successfully employed in many types of directed evolution experiments, including those involving carotenoid biosynthetic enzymes (1–3) and oxygenases (4–6).