Research Project
2024031
Using time-resolved digital imaging spectroscopy, we propose to implement high throughput solid phase filter assays for enzyme activity. This will allow us to screen large numbers (> 10(6)) of enzyme variants for improved activity directly in bacterial colonies. KAIROS' Digital Imaging Spectrophotometer (Colonylmager) will be adapted to the problem of imaging bacterial colony lifts on solid supports (i.e., filters) with colorimetric substrates for specific enzymes. A series of such images versus time will provide information on the substrate turnover rate by each colony. Phase I research and development will focus on improving the filter assay technique and on refining the imaging software used to obtain kinetic data. New software will analyze intra-feature pixel statistics, so as to minimize the distribution of rates obtained from a homogeneous population of colonies. This will enable the identification of colonies exhibiting substrate turnover rates marginally different from wild-type in subsequent mutagenesis experiments. Filter assays and imaging techniques will be made broadly applicable to common colorimetric indicators, beginning with hydrolytic substrates such as X-glu, X-gal, and BCIP. In Phase II, we plan to screen large numbers of enzyme variants for improved catalytic activity and adaptation to harsh conditions, such as high temperature, high/low pH, and organic solvents. We also plan to implement larger format scanners in place of the Colonylmager's CCD camera in order to achieve still higher throughput at lower cost. PROPOSED COMMERCIAL APPLICATION: KAIROS plans to include both commercial services and instrument sales: 1) Provide a service for rapid throughput solid phase analyses of mutagenized libraries for the purpose of identifying clones expressing enzymes with novel (new substrates, high temperature, organic solvents) or improved activity. 2) Market a low cost turn-key instrument to quantitate the results of solid phase enzymatic assays, especially in the emerging field of directed evolution.
