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J Biotechnol. 2017 Sep 20;258:25-32. doi: 10.1016/j.jbiotec.2017.05.006. Epub 2017 May 10.

A novel FbFP-based biosensor toolbox for sensitive in vivo determination of intracellular pH.

Author information

1
Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, D-52425 Jülich, Germany.
2
Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany; GO-Bio Projekt SenseUP, Forschungszentrum Jülich, D-52425 Jülich GmbH, Germany.
3
Institute of Complex Systems ICS-4: Cellular Biophysics, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany.
4
Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, D-52425 Jülich, Germany; Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany.
5
Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, D-52425 Jülich, Germany. Electronic address: t.drepper@fz-juelich.de.

Abstract

The intracellular pH is an important modulator of various bio(techno)logical processes such as enzymatic conversion of metabolites or transport across the cell membrane. Changes of intracellular pH due to altered proton distribution can thus cause dysfunction of cellular processes. Consequently, accurate monitoring of intracellular pH allows elucidating the pH-dependency of (patho)physiological and biotechnological processes. In this context, genetically encoded biosensors represent a powerful tool to determine intracellular pH values non-invasively and with high spatiotemporal resolution. We have constructed a toolbox of novel genetically encoded FRET-based pH biosensors (named Fluorescence Biosensors for pH or FluBpH) that utilizes the FMN-binding fluorescent protein EcFbFP as donor domain. In contrast to many fluorescent proteins of the GFP family, EcFbFP exhibits a remarkable tolerance towards acidic pH (pKa∼3.2). To cover the broad range of physiologically relevant pH values, three EYFP variants exhibiting pKa values of 5.7, 6.1 and 7.5 were used as pH-sensing FRET acceptor domains. The resulting biosensors FluBpH 5.7, FluBpH 6.1 and FluBpH 7.5 were calibrated in vitro and in vivo to accurately evaluate their pH indicator properties. To demonstrate the in vivo applicability of FluBpH, changes of intracellular pH were ratiometrically measured in E. coli cells during acid stress.

KEYWORDS:

Bacterial acid stress response; FRET biosensor; Flavin-binding fluorescent proteins (FbFP); Intracellular pH; Yellow fluorescent protein (YFP)

PMID:
28501596
DOI:
10.1016/j.jbiotec.2017.05.006
[Indexed for MEDLINE]

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