Tailored Methods to Develop High-Performance Chemigenetic Indicators

Fluorescent indicators are a powerful toolkit in biology to visualize spatiotemporal dynamics of analytes in live cells or tissues. Although indicators based on green fluorescent protein (GFP) and their analogs are well established, they have some limitations, such as low brightness in the red region of the spectrum. To address this issue, so-called chemigenetic indicators that are composed of a synthetic fluorescent ligand and a genetically encoded protein are attracting attention, which can in principle achieve substantially red-shifted fluorescence and higher brightness. Despite their promise, methods for the development of chemigenetic indicators remain at an early stage, and systematic methods to accelerate their optimization have not been well established. In this chapter, we describe detailed methods for developing high-performance chemigenetic indicators through high-throughput screening of protein libraries expressed in E. coli.