Design and spectral properties of the FRET-based oxygen biosensor FluBO. (A) Genetic construction of the 1194-bp biosensor gene. The YFP encoding DNA fragment (5'-end) was fused to the FbFP encoding fragment (3'-end) by a 36-bp oligonucleotide which codes for a peptide linker (L) containing a thrombin cleavage site. The restriction endonuclease cleavage sites used for the construction of the biosensor gene are shown. (B) Absorption and fluorescence spectra (λexc = 440 nm) of FbFP and YFP in phosphate buffer (pH 8.0, 10 mM NaCl). Absorption spectra are given considering the ratio of the absorption maxima of YFP (514 nm) and FbFP (450 nm) that corresponds to the ratio of the respective extinction coefficients (YFP : 72,000 cm-1M-1, FbFP: 12,500 cm-1M-1). Accordingly, the fluorescence spectra were adjusted considering the ratio of the respective fluorescence quantum yields (YFP : 0.76, FbFP: 0.39). The inset additionally shows the corresponding normalized FbFP and YFP fluorescence spectra. (C) Two-dimensional wavelength scans of FbFP and YFP. Purified proteins were adjusted to an absorption of 0.1 (λFbFP = 450 nm; λYFP = 514 nm) and fluorescence emission spectra (450 nm to 595 nm) were recorded upon increasing excitation wavelengths from 300 nm to 600 nm. The optimal excitation wavelength, where FbFP but not YFP showed bright fluorescence, is marked by dashed lines. (D) Emission spectrum of purified FluBO in comparison to the spectra of donor and acceptor fluorescent proteins FbFP and YFP at λexc = 380 nm; a.u.: arbitrary units. The three proteins were used in equimolar concentrations (approximately 1.5 μM) as judged by the overlap of their absorption spectra in the region > 500 nm (YFP and FluBO) and 320 to 380 nm (FbFP and FluBO).