Abstract
BackgroundMolecular 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.ResultsWe 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.ConclusionsFluBO 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.
Highlights
Molecular oxygen (O2) is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes
We constructed a recombinant gene (Figure 1A, Additional file 1) encoding the oxygen biosensor which consists of the N-terminal yellow fluorescent protein (YFP) acceptor domain and the C-terminal flavin-binding fluorescent protein (FbFP) donor domain interconnected by a small peptide linker with a thrombin protease cleavage site
FbFP exhibited its typical absorption spectrum ranging from near UV to blue light with lmax at 450 nm, which is characteristic for the flavin mononucleotide (FMN) chromophore [31]
Summary
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. The green fluorescent protein (GFP) and its variants can be applied as genetically encoded intracellular probes that are expressed and can be selectively targeted within defined cells and tissues. In this context, at least two ‘passive’ GFP-based oxygen sensors have been developed for estimating intracellular oxygen levels in E. coli. Oxygen sensitive photoactivation of GFPmediated red fluorescence was applied for in vivo imaging of oxygen in mammalian cells and organs [22,23,24]
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