Recent advances in the design of genetically encoded redox biosensors, such as redox-sensitive GFP (roGFP) have facilitated the real-time imaging of the intracellular redox potential in eukaryotic cells at high sensitivity and at spatiotemporal resolution. To increase the specificity of roGFP2 for the interaction with the glutathione (GSH)/ glutathione disulfide (GSSG) redox couple, roGFP2 has been fused to glutaredoxin (Grx) to construct the Grx-roGFP2 biosensor. We have previously designed the related Brx-roGFP2 redox biosensor for dynamic measurement of the bacillithiol redox potential (E-BSH) in the human pathogen Staphylococcus aureus. Here, we describe the detailed method for measurements of the oxidation degree (OxD) of the Brx-roGFP2 biosensor in S. aureus using the microplate reader. In particularly, we provide details for determination of the E-BSH changes during the growth and after oxidative stress. For future biosensor applications at the single cell level, we recommend the design of genome-encoded roGFP2 biosensors enabling stable expression and fluorescence in bacteria.
Brx-roGFP2 is specific for measurements of the bacillithiol redox potential in Staphylococcus aureus cells
Control samples for fully reduced and oxidized states of Brx-roGFP2 are required for calibration during OxD measurements
Easy to measure fluorescence excitation intensities at the 405 and 488 nm excitation maxima using microplate readers
