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Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Mazo-Vargas A, Park H, Aydin M, Buchler NE - Mol. Biol. Cell (2014)

Bottom Line: The photon flux per luciferase is significantly lower than that for fluorescent proteins.Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast.Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.

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Construction and design of multicolor luciferases. (A) We used gene synthesis (DNA 2.0, Menlo Park, CA) to build beetle luciferases—green from P. termitilluminans (Viviani et al., 1999b), yellow from P. pyralis (Fujii et al., 2007), and red from P. hirtus (Viviani et al., 1999a)—for expression in budding yeast. Beetle luciferase is an enzyme that catalyzes a two-step reaction that requires both ATP and O2 in addition to d-luciferin substrate. An N-terminal SV40 NLS was added between NcoI–NcoI to concentrate luciferase into the nucleus and improve signal-to-background ratio. The C-terminal SKL peroxisomal targeting motif (Leskinen et al., 2003) was removed and replaced by an ssrA degron between SpeI–SpeI for future use in an engineered ClpXP yeast strain (Grilly et al., 2007). The ClpXP strain has LacI-regulated expression of ClpX and ClpP, two subunits of a bacterial proteasome that recognizes a short amino acid sequence, ssrA. Any yeast protein in the engineered ClpXP strain that is fused to an ssrA tag will be conditionally degraded by the addition of isopropyl-β-d-thiogalactoside. (B) For comparison, we tested Promega CBG99 (green) and CBR (red) from P. plagiophthalamus and FLuc (yellow) from P. pyralis. We also tested Promega NLuc (blue), which is a bright marine luciferase that requires only O2 and furimazine (a coelenterazine analogue; Hall et al., 2012). Blue marine luciferases do not require ATP. All of these gene constructs were built to be modular and backward compatible with popular yeast enhanced fluorescent proteins (PacI-AscI; Sheff and Thorn, 2004) and yeast PEST degron (XhoI/BsrGI-AscI) derived from CLN2 gene (Mateus and Avery, 2000). (C) We built FLuc-yEVenus and FLuc-yEVenus-PEST fusion proteins to compare time-lapse luminescence and fluorescence microscopy directly.
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Figure 1: Construction and design of multicolor luciferases. (A) We used gene synthesis (DNA 2.0, Menlo Park, CA) to build beetle luciferases—green from P. termitilluminans (Viviani et al., 1999b), yellow from P. pyralis (Fujii et al., 2007), and red from P. hirtus (Viviani et al., 1999a)—for expression in budding yeast. Beetle luciferase is an enzyme that catalyzes a two-step reaction that requires both ATP and O2 in addition to d-luciferin substrate. An N-terminal SV40 NLS was added between NcoI–NcoI to concentrate luciferase into the nucleus and improve signal-to-background ratio. The C-terminal SKL peroxisomal targeting motif (Leskinen et al., 2003) was removed and replaced by an ssrA degron between SpeI–SpeI for future use in an engineered ClpXP yeast strain (Grilly et al., 2007). The ClpXP strain has LacI-regulated expression of ClpX and ClpP, two subunits of a bacterial proteasome that recognizes a short amino acid sequence, ssrA. Any yeast protein in the engineered ClpXP strain that is fused to an ssrA tag will be conditionally degraded by the addition of isopropyl-β-d-thiogalactoside. (B) For comparison, we tested Promega CBG99 (green) and CBR (red) from P. plagiophthalamus and FLuc (yellow) from P. pyralis. We also tested Promega NLuc (blue), which is a bright marine luciferase that requires only O2 and furimazine (a coelenterazine analogue; Hall et al., 2012). Blue marine luciferases do not require ATP. All of these gene constructs were built to be modular and backward compatible with popular yeast enhanced fluorescent proteins (PacI-AscI; Sheff and Thorn, 2004) and yeast PEST degron (XhoI/BsrGI-AscI) derived from CLN2 gene (Mateus and Avery, 2000). (C) We built FLuc-yEVenus and FLuc-yEVenus-PEST fusion proteins to compare time-lapse luminescence and fluorescence microscopy directly.

Mentions: We first designed and constructed green (GrLuc), yellow (YeLuc), and red (RdLuc) luciferase derived from beetles (Viviani et al., 1999a, b; Fujii et al., 2007). We fused an N-terminal nuclear-localization signal (NLS) to concentrate these luciferases into a smaller volume and spread the light signal across fewer pixels (Figure 1A). For comparison, we also tested commercial beetle luciferases (CBG99, CBR, FLuc) and a new marine luciferase (NLuc; Hall et al., 2012) from Promega (Madison, WI; Figure 1B). Each luciferase reporter was regulated by a methionine-repressible promoter (MET17) and integrated into the yeast genome either in single copy or multiple copies; see Materials and Methods. We measured the emission spectrum of our designed luciferases to confirm that they were consistent with their expected color; see Supplemental Figure S1.


Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Mazo-Vargas A, Park H, Aydin M, Buchler NE - Mol. Biol. Cell (2014)

Construction and design of multicolor luciferases. (A) We used gene synthesis (DNA 2.0, Menlo Park, CA) to build beetle luciferases—green from P. termitilluminans (Viviani et al., 1999b), yellow from P. pyralis (Fujii et al., 2007), and red from P. hirtus (Viviani et al., 1999a)—for expression in budding yeast. Beetle luciferase is an enzyme that catalyzes a two-step reaction that requires both ATP and O2 in addition to d-luciferin substrate. An N-terminal SV40 NLS was added between NcoI–NcoI to concentrate luciferase into the nucleus and improve signal-to-background ratio. The C-terminal SKL peroxisomal targeting motif (Leskinen et al., 2003) was removed and replaced by an ssrA degron between SpeI–SpeI for future use in an engineered ClpXP yeast strain (Grilly et al., 2007). The ClpXP strain has LacI-regulated expression of ClpX and ClpP, two subunits of a bacterial proteasome that recognizes a short amino acid sequence, ssrA. Any yeast protein in the engineered ClpXP strain that is fused to an ssrA tag will be conditionally degraded by the addition of isopropyl-β-d-thiogalactoside. (B) For comparison, we tested Promega CBG99 (green) and CBR (red) from P. plagiophthalamus and FLuc (yellow) from P. pyralis. We also tested Promega NLuc (blue), which is a bright marine luciferase that requires only O2 and furimazine (a coelenterazine analogue; Hall et al., 2012). Blue marine luciferases do not require ATP. All of these gene constructs were built to be modular and backward compatible with popular yeast enhanced fluorescent proteins (PacI-AscI; Sheff and Thorn, 2004) and yeast PEST degron (XhoI/BsrGI-AscI) derived from CLN2 gene (Mateus and Avery, 2000). (C) We built FLuc-yEVenus and FLuc-yEVenus-PEST fusion proteins to compare time-lapse luminescence and fluorescence microscopy directly.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230627&req=5

Figure 1: Construction and design of multicolor luciferases. (A) We used gene synthesis (DNA 2.0, Menlo Park, CA) to build beetle luciferases—green from P. termitilluminans (Viviani et al., 1999b), yellow from P. pyralis (Fujii et al., 2007), and red from P. hirtus (Viviani et al., 1999a)—for expression in budding yeast. Beetle luciferase is an enzyme that catalyzes a two-step reaction that requires both ATP and O2 in addition to d-luciferin substrate. An N-terminal SV40 NLS was added between NcoI–NcoI to concentrate luciferase into the nucleus and improve signal-to-background ratio. The C-terminal SKL peroxisomal targeting motif (Leskinen et al., 2003) was removed and replaced by an ssrA degron between SpeI–SpeI for future use in an engineered ClpXP yeast strain (Grilly et al., 2007). The ClpXP strain has LacI-regulated expression of ClpX and ClpP, two subunits of a bacterial proteasome that recognizes a short amino acid sequence, ssrA. Any yeast protein in the engineered ClpXP strain that is fused to an ssrA tag will be conditionally degraded by the addition of isopropyl-β-d-thiogalactoside. (B) For comparison, we tested Promega CBG99 (green) and CBR (red) from P. plagiophthalamus and FLuc (yellow) from P. pyralis. We also tested Promega NLuc (blue), which is a bright marine luciferase that requires only O2 and furimazine (a coelenterazine analogue; Hall et al., 2012). Blue marine luciferases do not require ATP. All of these gene constructs were built to be modular and backward compatible with popular yeast enhanced fluorescent proteins (PacI-AscI; Sheff and Thorn, 2004) and yeast PEST degron (XhoI/BsrGI-AscI) derived from CLN2 gene (Mateus and Avery, 2000). (C) We built FLuc-yEVenus and FLuc-yEVenus-PEST fusion proteins to compare time-lapse luminescence and fluorescence microscopy directly.
Mentions: We first designed and constructed green (GrLuc), yellow (YeLuc), and red (RdLuc) luciferase derived from beetles (Viviani et al., 1999a, b; Fujii et al., 2007). We fused an N-terminal nuclear-localization signal (NLS) to concentrate these luciferases into a smaller volume and spread the light signal across fewer pixels (Figure 1A). For comparison, we also tested commercial beetle luciferases (CBG99, CBR, FLuc) and a new marine luciferase (NLuc; Hall et al., 2012) from Promega (Madison, WI; Figure 1B). Each luciferase reporter was regulated by a methionine-repressible promoter (MET17) and integrated into the yeast genome either in single copy or multiple copies; see Materials and Methods. We measured the emission spectrum of our designed luciferases to confirm that they were consistent with their expected color; see Supplemental Figure S1.

Bottom Line: The photon flux per luciferase is significantly lower than that for fluorescent proteins.Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast.Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.

Show MeSH
Related in: MedlinePlus