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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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Optimization of pH and substrate concentration in the extracellular medium. We used multicopy strains AMV104, AMV68, AMV69, AMV45, AMV152, AMV151, AMV16, and AMV41, which have a MET17 promoter driving different luciferase reporters; see Table 3. We also included the parental strain (MMY116-2C) as a negative control. Strains were induced overnight and diluted into fresh medium in the morning. Cell cultures were then grown at 30°C to OD660 ≈ 0.2 before starting measurements. Growth medium was synthetic complete methionine drop-out medium with 2% d-glucose (SCD–Met). The bulk luminescence was measured using a 96-well plate assay with a Wallac Victor 3 plate reader. Error bars represent the SD of three technical replicates. (A) We varied the d-luciferin and furimazine concentrations at constant pH values (3.8 and 6.0, respectively). (B) We varied the pH of the growth medium at constant d-luciferin (100 μM) or furimazine (20 μM) concentrations. On the basis of these results, we fixed the pH (3.8), d-luciferin (100 μM), and furimazine (20 μM) for all subsequent luminescence experiments. (C) We measured OD660 every 30 min with a spectrophotometer to quantify strain growth rates in SCD-Met at 30°C. Thin, dotted lines are the 95% confidence interval of the best exponential fit. Both single-copy and multicopy Nluc exhibited slower growth rates than the parental strain.
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Figure 2: Optimization of pH and substrate concentration in the extracellular medium. We used multicopy strains AMV104, AMV68, AMV69, AMV45, AMV152, AMV151, AMV16, and AMV41, which have a MET17 promoter driving different luciferase reporters; see Table 3. We also included the parental strain (MMY116-2C) as a negative control. Strains were induced overnight and diluted into fresh medium in the morning. Cell cultures were then grown at 30°C to OD660 ≈ 0.2 before starting measurements. Growth medium was synthetic complete methionine drop-out medium with 2% d-glucose (SCD–Met). The bulk luminescence was measured using a 96-well plate assay with a Wallac Victor 3 plate reader. Error bars represent the SD of three technical replicates. (A) We varied the d-luciferin and furimazine concentrations at constant pH values (3.8 and 6.0, respectively). (B) We varied the pH of the growth medium at constant d-luciferin (100 μM) or furimazine (20 μM) concentrations. On the basis of these results, we fixed the pH (3.8), d-luciferin (100 μM), and furimazine (20 μM) for all subsequent luminescence experiments. (C) We measured OD660 every 30 min with a spectrophotometer to quantify strain growth rates in SCD-Met at 30°C. Thin, dotted lines are the 95% confidence interval of the best exponential fit. Both single-copy and multicopy Nluc exhibited slower growth rates than the parental strain.

Mentions: We then used a 96-well plate bulk assay with a Wallac Victor 3 luminometer (PerkinElmer-Cetus, Waltham, MA) to optimize conditions (substrates, pH) for in vivo yeast luminescence. Beetle luciferases require d-luciferin, ATP, and oxygen as substrates, whereas NLuc only requires furimazine (a coelenterazine analogue) and oxygen. We expected log-phase cells to maintain their ATP at high concentrations. However, in vivo oxygen, d-luciferin, and furimazine could be limiting because they must diffuse across the lipid bilayer from the external medium (Wood and DeLuca, 1987; Vieites et al., 1994). We determined that 200 μM d-luciferin and 50 μM furimazine were saturating for in vivo luminescence (Figure 2A). We further boosted the in vivo luminescence of beetle luciferases ∼30-fold by lowering the pH of the growth medium (Figure 2B). The luminescence increased because of the low extracellular pH, which reduced the charge of d-luciferin (pKa = 2.9) and improved its diffusion across the lipid bilayer (Wood and DeLuca, 1987; Vieites et al., 1994). We verified that our yeast strains grew well at lower pH (3.8) and that the luciferases were not cytotoxic at high copy number (Figure 2C). The only exception is NLuc, which was already cytotoxic as a single-copy integrant (Figure 2C).


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)

Optimization of pH and substrate concentration in the extracellular medium. We used multicopy strains AMV104, AMV68, AMV69, AMV45, AMV152, AMV151, AMV16, and AMV41, which have a MET17 promoter driving different luciferase reporters; see Table 3. We also included the parental strain (MMY116-2C) as a negative control. Strains were induced overnight and diluted into fresh medium in the morning. Cell cultures were then grown at 30°C to OD660 ≈ 0.2 before starting measurements. Growth medium was synthetic complete methionine drop-out medium with 2% d-glucose (SCD–Met). The bulk luminescence was measured using a 96-well plate assay with a Wallac Victor 3 plate reader. Error bars represent the SD of three technical replicates. (A) We varied the d-luciferin and furimazine concentrations at constant pH values (3.8 and 6.0, respectively). (B) We varied the pH of the growth medium at constant d-luciferin (100 μM) or furimazine (20 μM) concentrations. On the basis of these results, we fixed the pH (3.8), d-luciferin (100 μM), and furimazine (20 μM) for all subsequent luminescence experiments. (C) We measured OD660 every 30 min with a spectrophotometer to quantify strain growth rates in SCD-Met at 30°C. Thin, dotted lines are the 95% confidence interval of the best exponential fit. Both single-copy and multicopy Nluc exhibited slower growth rates than the parental strain.
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Figure 2: Optimization of pH and substrate concentration in the extracellular medium. We used multicopy strains AMV104, AMV68, AMV69, AMV45, AMV152, AMV151, AMV16, and AMV41, which have a MET17 promoter driving different luciferase reporters; see Table 3. We also included the parental strain (MMY116-2C) as a negative control. Strains were induced overnight and diluted into fresh medium in the morning. Cell cultures were then grown at 30°C to OD660 ≈ 0.2 before starting measurements. Growth medium was synthetic complete methionine drop-out medium with 2% d-glucose (SCD–Met). The bulk luminescence was measured using a 96-well plate assay with a Wallac Victor 3 plate reader. Error bars represent the SD of three technical replicates. (A) We varied the d-luciferin and furimazine concentrations at constant pH values (3.8 and 6.0, respectively). (B) We varied the pH of the growth medium at constant d-luciferin (100 μM) or furimazine (20 μM) concentrations. On the basis of these results, we fixed the pH (3.8), d-luciferin (100 μM), and furimazine (20 μM) for all subsequent luminescence experiments. (C) We measured OD660 every 30 min with a spectrophotometer to quantify strain growth rates in SCD-Met at 30°C. Thin, dotted lines are the 95% confidence interval of the best exponential fit. Both single-copy and multicopy Nluc exhibited slower growth rates than the parental strain.
Mentions: We then used a 96-well plate bulk assay with a Wallac Victor 3 luminometer (PerkinElmer-Cetus, Waltham, MA) to optimize conditions (substrates, pH) for in vivo yeast luminescence. Beetle luciferases require d-luciferin, ATP, and oxygen as substrates, whereas NLuc only requires furimazine (a coelenterazine analogue) and oxygen. We expected log-phase cells to maintain their ATP at high concentrations. However, in vivo oxygen, d-luciferin, and furimazine could be limiting because they must diffuse across the lipid bilayer from the external medium (Wood and DeLuca, 1987; Vieites et al., 1994). We determined that 200 μM d-luciferin and 50 μM furimazine were saturating for in vivo luminescence (Figure 2A). We further boosted the in vivo luminescence of beetle luciferases ∼30-fold by lowering the pH of the growth medium (Figure 2B). The luminescence increased because of the low extracellular pH, which reduced the charge of d-luciferin (pKa = 2.9) and improved its diffusion across the lipid bilayer (Wood and DeLuca, 1987; Vieites et al., 1994). We verified that our yeast strains grew well at lower pH (3.8) and that the luciferases were not cytotoxic at high copy number (Figure 2C). The only exception is NLuc, which was already cytotoxic as a single-copy integrant (Figure 2C).

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