title: Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.
abstract: Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET). Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM). For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV)-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.
authors.affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG Groningen, The Netherlands.
authors.forename: Ruud G J
authors.initials: RG
authors.lastname: Detert Oude Weme
authors.affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG Groningen, The Netherlands.
authors.forename: Ákos T
authors.initials: ÁT
authors.lastname: Kovács
authors.affiliation: Lambert Instruments B.V., Oosteinde 16, 9301 ZP Roden, The Netherlands.
authors.forename: Sander J G
authors.initials: SJ
authors.lastname: de Jong
authors.affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG Groningen, The Netherlands.
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: Veening
authors.affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG Groningen, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Siebring
authors.affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG Groningen, The Netherlands.
authors.forename: Oscar P
authors.initials: OP
authors.lastname: Kuipers
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data
chemical_list: D049452:Green Fluorescent Proteins