Fig. 1.

Construction of an oxygen-sensitive fluorescent protein-based biosensor. (A) Schematic representations of full-length Sima protein (dHIF1-alpha; top), with its O₂-dependent degradation domain (ODD; aa 673-895), of the GFP-ODD fusion protein (middle), and of the GFP-ODD construct with the P850 residue mutated into alanine [GFP-ODD(P850A); bottom]. (B) Embryos showing the sensitivity of GFP-ODD to O₂ tension. GFP-ODD signals (top panels) were reduced in embryos incubated at 21% O₂ compared to embryos incubated at 5% O₂. The P850A mutation renders the GFP-ODD construct largely insensitive to changing O₂ levels (bottom panels). Scale bar: 100 µm. (C) Schematic representation of the ratiometric sensor design. The red fluorescence from a control protein (mRFP-nls) remains relatively constant under changing O₂ concentrations, whereas GFP-ODD is sensitive to O₂ levels, providing a measure of the cellular hypoxia response state.