A Hypersensitive Genetically Encoded Fluorescent Indicator (roGFP2-Prx1) Enables Continuous Measurement of Intracellular H2O2 during Cell Micro-cultivation
Artikel i vetenskaplig tidskrift, 2022

Hydrogen peroxide (H2O2) 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 H2O2 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 H2O2 levels in the budding yeast Saccharomyces cerevisiae 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 H2O2, ultimately resulting in a GFP signal being emitted by the sensor. Here, we apply this biosensor to study cytosolic H2O2 levels in S. cerevisiae strains with and without recombinant protein production.

Biosensor

Redox

Oxidative stress

Saccharomyces cerevisiae

Genetically encoded fluorescent indicator

In vivo measurement

H2O2

Oxidants

Yeast

GEFI

Författare

Veronica Gast

Chalmers, Biologi och bioteknik, Systembiologi

Verena Siewers

Chalmers, Biologi och bioteknik, Systembiologi

Mikael Molin

Chalmers, Biologi och bioteknik, Systembiologi

Bio-protocol

23318325 (eISSN)

Vol. 12 3 e4317

Väteperoxidsignaleringens och peroxiredoxins roll i åldrandet och åldersrelaterad nedgång i protein homeostasen

Vetenskapsrådet (VR) (2020-05422), 2021-01-01 -- 2024-12-31.

Ämneskategorier

Cellbiologi

Biokemi och molekylärbiologi

Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

DOI

10.21769/BioProtoc.4317

PubMed

35284600

Mer information

Senast uppdaterat

2022-04-21