A Hypersensitive Genetically Encoded Fluorescent Indicator (roGFP2-Prx1) Enables Continuous Measurement of Intracellular HO during Cell Micro-cultivation.

Hydrogen peroxide (HO) is a toxic oxidant produced as a byproduct of several biological processes. At too high levels of hydrogen peroxide cells will experience oxidative stress, leading to a cellular response to decrease its levels and to protect the cells. Previously, methods used to study and quantify intracellular HO have been limited by both sensitivity and specificity. However, an increasing number of genetically encoded fluorescent indicators (GEFIs) are becoming available, which can specifically detect low levels of intracellular hydrogen peroxide. In this study, we use such a biosensor designed to monitor cytosolic HO levels in the budding yeast during continuous cultivation and in the absence of a fluorescence microscope. The fluorescent biosensor contains a peroxiredoxin protein fused to an engineered GFP molecule expressed from a commonly used yeast plasmid (pRS416-TEF1). The peroxiredoxin-based fluorescent indicator reduces HO, ultimately resulting in a GFP signal being emitted by the sensor. Here, we apply this biosensor to study cytosolic HO levels in strains with and without recombinant protein production.