Biophotonics: Directed Evolution of GFP-based Probes for Glucose

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0086920WoodburyThe applicants proposed to develop a glucose biosensor using a directed evolution approach based on the green fluorescent protein (GFP). A library of DNA sequences encoding glucose binding motifs will be inserted into an appropriate position in the gene for GFP and directed evolution methods will be applied to generate a bioprobe that fluoresces much more brightly when bound to glucose than in the unbound state. High throughput screening of fluorescence from microcolonies will be performed using a high resolution CCD camera. The selection of individual microcolonies will then be performed using a novel photolithography based approach in which a digital light processor is used to image UV light onto bacterial plates with high resolution (patterned cell growth). The remaining live bacteria will be grown, plasmids isolated and subsequent rounds of directed evolution will be initiated using a combination of error prone PCR and DNA shuffling to generate additional variation in the gene sequence. Screening of colonies in later stages of the directed evolution will also take advantage of fluorescence lifetime imaging using a scanning confocal microscope and a mode-locked laser. This will help to discriminate between variations in the glucose binding constant and changes in the GFP fluorescence lifetime upon binding. GFP-based probes generated in this way have the advantage that they can either be expressed in E. coli, purified and used directly for biosensor applications without modification, or they can be expressed in modified host cells generating a living biosensor that constantly replenishes the sensor molecule. This procedure for generating GFP-based bioprobes is general and can be applied to a large number of potential target molecules.