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Open Access Methodology article

Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

Janko Potzkei1, Martin Kunze2, Thomas Drepper1*, Thomas Gensch3, Karl-Erich Jaeger1* and Jochen Büchs2

Author Affiliations

1 Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Juelich Research Center, Wilhelm-Johnen-Straße, D-52425 Juelich, Germany

2 AVT Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany

3 Institute of Complex Systems-Cellular Biophysics (ICS-4), Juelich Research Center, Wilhelm-Johnen-Straße, D-52425 Juelich, Germany

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BMC Biology 2012, 10:28  doi:10.1186/1741-7007-10-28

Published: 22 March 2012

Additional files

Additional file 1:

FluBO nucleotide and amino acid sequence. Yellow, YFP sequence; black, linker sequence; green, FbFP sequence; grey, His6 tag sequence.

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Additional file 2:

Fluorescence spectra of FluBO before and after proteolysis with thrombin. Fluorescence spectra of FluBO before and after proteolysis with thrombin. The fluorescence emission spectra of purified FPs (5 μM) were recorded at an excitation wavelength of 380 nm. Complete thrombin-catalyzed proteolysis of FluBO was demonstrated by SDS-PAGE (inset), purified FPs were used as standards.

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Additional file 3:

Sensitivity of used YFP towards pH. The figure shows the normalized YFP fluorescence intensity at increasing pH values. Purified YFP was adjusted to an absorption of 0.05. The effect of the pH value on YFP fluorescence was measured in citric acid/disodium phosphate buffer with pH values ranging form 4.0 to 7.0. The pKa value is marked by doted lines.

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Additional file 4:

Data from parallel fermentation experiments. The figure shows the individual results from three independent batch cultivation experiments using the BioLector. The development of biomass (black), dissolved oxygen tension (blue) and cyan-to-yellow fluorescent ratio (emYFP/emFbFP) of the FRET-based oxygen biosensor (red) was online-monitored in each well of the flowerplate.

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