Optimization of a Genetically Encoded Optical Zinc Probe
Biological investigations into the cell have been amplified with fluorescent sensors. Through the use of a novel genetically encoded optical sensor, the biological functions of intracellular labile Zn2+ can be detected at the nanomolar range within live cells. Using a single-fluorescent protein-based platform, the GZnP family of Zn2+ sensors was developed. Although the existing sensors allow for Zn2+ dynamics to be monitored within targeted subcellular components, the dynamic range, brightness, and kinetics can be improved to generate sensors with a higher sensitivity. To further optimize the sensitivity, a high throughput cell lysate screening assay can be utilized to identify new sensor variants with improved characteristics. In this work, we created and evaluated a series of sensor libraries comprised of mutations in order to identify new sensor variants with improved characteristics. This characterization was achieved through confocal microscopy. Optimization of the GZnP sensors serves to guide the visualization of Zn2+ dynamics and advancing our understanding of cellular Zn2+ function.