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BMC Biol. 2012; 10: 28.
Published online Mar 22, 2012. doi:  10.1186/1741-7007-10-28
PMCID: PMC3364895
Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor
Janko Potzkei,#1 Martin Kunze,#2 Thomas Drepper,corresponding author#1 Thomas Gensch,3 Karl-Erich Jaeger,corresponding author1 and Jochen Büchs2
1Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Juelich Research Center, Wilhelm-Johnen-Straße, D-52425 Juelich, Germany
2AVT Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany
3Institute of Complex Systems-Cellular Biophysics (ICS-4), Juelich Research Center, Wilhelm-Johnen-Straße, D-52425 Juelich, Germany
corresponding authorCorresponding author.
#Contributed equally.
Janko Potzkei: j.potzkei/at/fz-juelich.de; Martin Kunze: Martin.Kunze/at/avt.rwth-aachen.de; Thomas Drepper: t.drepper/at/fz-juelich.de; Thomas Gensch: t.gensch/at/fz-juelich.de; Karl-Erich Jaeger: k.-e.jaeger/at/fz-juelich.de; Jochen Büchs: jochen.buechs/at/avt.rwth-aachen.de
Received December 16, 2011; Accepted March 22, 2012.
Abstract
Background
Molecular oxygen (O2) is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues.
Results
We developed a genetically encoded Förster resonance energy transfer (FRET)-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen) consists of the yellow fluorescent protein (YFP) that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP). Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation.
Conclusions
FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (patho)physiological processes in living cells.
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