Limits...
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.

Show MeSH

Related in: MedlinePlus

Luminescence output of different multicolor luciferases integrated in yeast chromosome as (A) a single copy or (B) multiple copies. Single-copy strains were AMV70, AMV72, AMV71, AMV54, AMV152-13, AMV151-18, and AMV16, and “bright,” multicopy strains were AMV104, AMV68, AMV69, AMV45, AMV152-03, and AMV151-08; see Table 3. All strains have a MET17 promoter driving different luciferase reporters. We also included the parental strain (MMY116-2C) as a negative control. We used a 96-well plate assay, and error bars represent the SD of three technical replicates. At time t = 0 min, 100 μl of log-phase (OD660 = 0.1) yeast pregrown at 30°C in SCD–Met were inoculated in fresh, identical medium with 100 μM d-luciferin at pH 3.8 or 20 μM furimazine at pH 6.0. Time-lapse luminescence microscopy at subminute time resolution was successful for those strains above the horizontal, dashed line at 4 × 104 luminescence units. With the exception of CBR, commercial luciferases are bright enough at single copy for subminute time-lapse luminescence microscopy in budding yeast.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230627&req=5

Figure 3: Luminescence output of different multicolor luciferases integrated in yeast chromosome as (A) a single copy or (B) multiple copies. Single-copy strains were AMV70, AMV72, AMV71, AMV54, AMV152-13, AMV151-18, and AMV16, and “bright,” multicopy strains were AMV104, AMV68, AMV69, AMV45, AMV152-03, and AMV151-08; see Table 3. All strains have a MET17 promoter driving different luciferase reporters. We also included the parental strain (MMY116-2C) as a negative control. We used a 96-well plate assay, and error bars represent the SD of three technical replicates. At time t = 0 min, 100 μl of log-phase (OD660 = 0.1) yeast pregrown at 30°C in SCD–Met were inoculated in fresh, identical medium with 100 μM d-luciferin at pH 3.8 or 20 μM furimazine at pH 6.0. Time-lapse luminescence microscopy at subminute time resolution was successful for those strains above the horizontal, dashed line at 4 × 104 luminescence units. With the exception of CBR, commercial luciferases are bright enough at single copy for subminute time-lapse luminescence microscopy in budding yeast.

Mentions: We also compared the relative brightness of different luciferases. To this end, we integrated a single gene copy of each luciferase into the same target locus. Our beetle luciferases GrLuc and YeLuc were ∼10-fold brighter than RdLuc (Figure 3A). This same color relationship persists for Promega beetle luciferases CBG99 (green), FLuc (yellow), and CBR (red). All of the commercial luciferases were at least ∼10-fold brighter (Figure 3A) than our designed luciferases. NLuc was the brightest. Unfortunately, furimazine was insoluble in aqueous solution and precipitated out, which made NLuc incompatible with long-term, live-cell time-lapse luminescence microscopy.


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)

Luminescence output of different multicolor luciferases integrated in yeast chromosome as (A) a single copy or (B) multiple copies. Single-copy strains were AMV70, AMV72, AMV71, AMV54, AMV152-13, AMV151-18, and AMV16, and “bright,” multicopy strains were AMV104, AMV68, AMV69, AMV45, AMV152-03, and AMV151-08; see Table 3. All strains have a MET17 promoter driving different luciferase reporters. We also included the parental strain (MMY116-2C) as a negative control. We used a 96-well plate assay, and error bars represent the SD of three technical replicates. At time t = 0 min, 100 μl of log-phase (OD660 = 0.1) yeast pregrown at 30°C in SCD–Met were inoculated in fresh, identical medium with 100 μM d-luciferin at pH 3.8 or 20 μM furimazine at pH 6.0. Time-lapse luminescence microscopy at subminute time resolution was successful for those strains above the horizontal, dashed line at 4 × 104 luminescence units. With the exception of CBR, commercial luciferases are bright enough at single copy for subminute time-lapse luminescence microscopy in budding yeast.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230627&req=5

Figure 3: Luminescence output of different multicolor luciferases integrated in yeast chromosome as (A) a single copy or (B) multiple copies. Single-copy strains were AMV70, AMV72, AMV71, AMV54, AMV152-13, AMV151-18, and AMV16, and “bright,” multicopy strains were AMV104, AMV68, AMV69, AMV45, AMV152-03, and AMV151-08; see Table 3. All strains have a MET17 promoter driving different luciferase reporters. We also included the parental strain (MMY116-2C) as a negative control. We used a 96-well plate assay, and error bars represent the SD of three technical replicates. At time t = 0 min, 100 μl of log-phase (OD660 = 0.1) yeast pregrown at 30°C in SCD–Met were inoculated in fresh, identical medium with 100 μM d-luciferin at pH 3.8 or 20 μM furimazine at pH 6.0. Time-lapse luminescence microscopy at subminute time resolution was successful for those strains above the horizontal, dashed line at 4 × 104 luminescence units. With the exception of CBR, commercial luciferases are bright enough at single copy for subminute time-lapse luminescence microscopy in budding yeast.
Mentions: We also compared the relative brightness of different luciferases. To this end, we integrated a single gene copy of each luciferase into the same target locus. Our beetle luciferases GrLuc and YeLuc were ∼10-fold brighter than RdLuc (Figure 3A). This same color relationship persists for Promega beetle luciferases CBG99 (green), FLuc (yellow), and CBR (red). All of the commercial luciferases were at least ∼10-fold brighter (Figure 3A) than our designed luciferases. NLuc was the brightest. Unfortunately, furimazine was insoluble in aqueous solution and precipitated out, which made NLuc incompatible with long-term, live-cell time-lapse luminescence microscopy.

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