During Phase 1, a prototype MicroColonylmager (MCI) and colorimetric solid phase assays were developed to screen bacterial libraries expressing mutagenized enzymes for enhanced activity. This high-throughput assay can detect less than a 3-fold difference in enzyme rates within microcolonies grown at a nearly confluent density of 500 colonies per cm2 on a 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 current liquid phase assays. By simultaneously assaying two different substrates tagged with spectrally distinct chromogenic reporters, we have successfully used this technique to change the substrate specificity of a beta-glucosidase from glucoside to its epimer, galactoside. In Phase 2, we will refine MCI hardware and software to enable high-throughput screening of enzyme libraries by time course analyses of single-pixels, using either absorption, fluorescence or FRET. Membrane disk size and colony density will be increased to image approximately 50,000 microcolonies simultaneously, while decreasing substrate volumes to approximately 10 nanoliters per measurement. Concurrent with instrument development, we will demonstrate the commercial value of this technology by screening libraries undergoing directed evolution. This technology should lead to the isolation of new enzyme activities that are useful in the synthesis of various substances, including pharmaceuticals.
A MicroColonylmeger (MCI) and solid phase assay for high-throughput screening of libraries expressing mutagenized enzymes will be developed and applied to the massively parallel screening of enzymes undergoing directed evolution in E.coli. We will demonstrate this new technology on four different enzyme development projects to isolate variant enzymes having activities that are commercially useful for the organic/ biochemical synthesis of various substances, including chiral pharmaceuticals.
