Limits...
Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

Detert Oude Weme RG, Kovács ÁT, de Jong SJ, Veening JW, Siebring J, Kuipers OP - PLoS ONE (2015)

Bottom Line: 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.This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG Groningen, The Netherlands.

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.

Show MeSH

Related in: MedlinePlus

(A) The amyE integration vector pDOW23 with FRET-pair GFP-tagRFP.(B) Schematic representation of two fluorescent proteins and the linker containing the TEV-protease recognition site (ENLYFQG). (C) A Western Blot to show the cleaving of coupled fluorophores by the TEV-protease. GFP protein was visualized by chemiluminescence with GFP-antibodies. The lanes contain cell free extract from the following strains: lane 1, DOW05 (thrC::Pxyl-tev amyE::gfp), lane 2, DOW13 (thrC::Pxyl-tev amyE::tagRFP), lane 3, DOW23 (thrC::Pxyl-tev amyE::gfp-tagRFP) from a culture without induction of the tev protease gene and lane 4, DOW23 (thrC::Pxyl-tev amyE::gfp-tagRFP) in which the tev protease gene was induced with 1% (w/v) xylose. Predicted sizes for GFP and tagRFP monomer are 27 kDa, and the complex 55 kDa.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4401445&req=5

pone.0123239.g002: (A) The amyE integration vector pDOW23 with FRET-pair GFP-tagRFP.(B) Schematic representation of two fluorescent proteins and the linker containing the TEV-protease recognition site (ENLYFQG). (C) A Western Blot to show the cleaving of coupled fluorophores by the TEV-protease. GFP protein was visualized by chemiluminescence with GFP-antibodies. The lanes contain cell free extract from the following strains: lane 1, DOW05 (thrC::Pxyl-tev amyE::gfp), lane 2, DOW13 (thrC::Pxyl-tev amyE::tagRFP), lane 3, DOW23 (thrC::Pxyl-tev amyE::gfp-tagRFP) from a culture without induction of the tev protease gene and lane 4, DOW23 (thrC::Pxyl-tev amyE::gfp-tagRFP) in which the tev protease gene was induced with 1% (w/v) xylose. Predicted sizes for GFP and tagRFP monomer are 27 kDa, and the complex 55 kDa.

Mentions: The aim of this work was to identify the best FRET-pair and to perform FRET at the single cell level in B. subtilis using fluorescence microscopy. Therefore, the suitability of various fluorescent proteins (FPs) for FRET purposes in B. subtilis was tested by expressing them pairwise and covalently linked. The FPs tested here were Cerulean (a cyan FP [25]), Venus (a yellow FP [26]), and sfGFP(Sp) [27] as donor and tagRFP (a red-orange FP, Evrogen), mCherry (a red FP [28]), and mKate2 (a far-red FP, Evrogen) as acceptor. The respective genes were cloned in the amyE locus of B. subtilis under control of the IPTG-inducible Phyper-spank promoter, by using the newly constructed amyE integration vector pDOW (Fig 1), which allows for efficient BglBrick [23] assembly (Fig 2A).


Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

Detert Oude Weme RG, Kovács ÁT, de Jong SJ, Veening JW, Siebring J, Kuipers OP - PLoS ONE (2015)

(A) The amyE integration vector pDOW23 with FRET-pair GFP-tagRFP.(B) Schematic representation of two fluorescent proteins and the linker containing the TEV-protease recognition site (ENLYFQG). (C) A Western Blot to show the cleaving of coupled fluorophores by the TEV-protease. GFP protein was visualized by chemiluminescence with GFP-antibodies. The lanes contain cell free extract from the following strains: lane 1, DOW05 (thrC::Pxyl-tev amyE::gfp), lane 2, DOW13 (thrC::Pxyl-tev amyE::tagRFP), lane 3, DOW23 (thrC::Pxyl-tev amyE::gfp-tagRFP) from a culture without induction of the tev protease gene and lane 4, DOW23 (thrC::Pxyl-tev amyE::gfp-tagRFP) in which the tev protease gene was induced with 1% (w/v) xylose. Predicted sizes for GFP and tagRFP monomer are 27 kDa, and the complex 55 kDa.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401445&req=5

pone.0123239.g002: (A) The amyE integration vector pDOW23 with FRET-pair GFP-tagRFP.(B) Schematic representation of two fluorescent proteins and the linker containing the TEV-protease recognition site (ENLYFQG). (C) A Western Blot to show the cleaving of coupled fluorophores by the TEV-protease. GFP protein was visualized by chemiluminescence with GFP-antibodies. The lanes contain cell free extract from the following strains: lane 1, DOW05 (thrC::Pxyl-tev amyE::gfp), lane 2, DOW13 (thrC::Pxyl-tev amyE::tagRFP), lane 3, DOW23 (thrC::Pxyl-tev amyE::gfp-tagRFP) from a culture without induction of the tev protease gene and lane 4, DOW23 (thrC::Pxyl-tev amyE::gfp-tagRFP) in which the tev protease gene was induced with 1% (w/v) xylose. Predicted sizes for GFP and tagRFP monomer are 27 kDa, and the complex 55 kDa.
Mentions: The aim of this work was to identify the best FRET-pair and to perform FRET at the single cell level in B. subtilis using fluorescence microscopy. Therefore, the suitability of various fluorescent proteins (FPs) for FRET purposes in B. subtilis was tested by expressing them pairwise and covalently linked. The FPs tested here were Cerulean (a cyan FP [25]), Venus (a yellow FP [26]), and sfGFP(Sp) [27] as donor and tagRFP (a red-orange FP, Evrogen), mCherry (a red FP [28]), and mKate2 (a far-red FP, Evrogen) as acceptor. The respective genes were cloned in the amyE locus of B. subtilis under control of the IPTG-inducible Phyper-spank promoter, by using the newly constructed amyE integration vector pDOW (Fig 1), which allows for efficient BglBrick [23] assembly (Fig 2A).

Bottom Line: 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.This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG Groningen, The Netherlands.

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.

Show MeSH
Related in: MedlinePlus