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NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level.

Knudsen JD, Carlquist M, Gorwa-Grauslund M - AMB Express (2014)

Bottom Line: The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in the gpd1Δgpd2Δ strain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH.In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression.The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Applied Microbiology, Department of Chemistry, Lund University, Lund, 221 00, SE, Sweden.

ABSTRACT
A reporter system was constructed to measure perturbations in the NADH/NAD(+) co-factor balance in yeast, by using the green fluorescent protein gene under the control of the GPD2 promoter that is induced under conditions of excess of NADH. High fluorescence levels were obtained in a glycerol 3-phosphate dehydrogenase double deletion strain (gpd1Δgpd2Δ), which is deficient in the ability to regenerate NAD(+) via glycerol formation. The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in the gpd1Δgpd2Δ strain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH. Addition of acetoin during cell proliferation under oxygen-limited conditions resulted in a more than 2-fold decrease in mean fluorescence intensity as compared to the control experiment. Strains carrying XRs with different selectivities for NADH could be distinguished at the single cell level, so that the XR with the highest selectivity for NADH displayed the lowest fluorescence. In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression. The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells.

No MeSH data available.


Related in: MedlinePlus

Fluorescence plots after 24 hours of growth. (A) Histograms of the FI of the control strains carrying no XR “red dash”, wtXR “blue dash” or mutXR “green dash”). (B) Histogram of the FI of the gpd1Δ gpd2Δ strains carrying no XR (red dash), wtXR (blue dash) or mutXR (green dash). Legend TMB4140: control, no XR; TMB4141: control, wtXR; TMB4143: control, mutXR; TMB4144: gpd1Δgpd2Δ, no XR;TMB4145: gpd1Δgpd2Δ, wtXR; TMB4147:gpd1Δgpd2Δ, mutXR.
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Figure 5: Fluorescence plots after 24 hours of growth. (A) Histograms of the FI of the control strains carrying no XR “red dash”, wtXR “blue dash” or mutXR “green dash”). (B) Histogram of the FI of the gpd1Δ gpd2Δ strains carrying no XR (red dash), wtXR (blue dash) or mutXR (green dash). Legend TMB4140: control, no XR; TMB4141: control, wtXR; TMB4143: control, mutXR; TMB4144: gpd1Δgpd2Δ, no XR;TMB4145: gpd1Δgpd2Δ, wtXR; TMB4147:gpd1Δgpd2Δ, mutXR.

Mentions: The population histogram, i.e. the amount of single cells displaying a given FI, also clearly showed differences between the strains (Figures 5A, B). After 24 hours, the different populations displayed significantly different FIs, and were thus easily distinguished in an overlay plot. This was the case both in the control (Figure 5A) and in the gpd1Δ gpd2Δ (Figure 5B) strains, with mean FIs given in channel number units of 204 (control, no XR), 160 (control, wtXR), 70 (control, mutXR) or 325 (gpd1Δ gpd2Δ, no XR), 250 (gpd1Δ gpd2Δ, wtXR) and 171 (gpd1Δ gpd2Δ, mutXR), respectively. Some overlap between strains was observed in both strain backgrounds, but it was more pronounced for the wild type strains than for the deletions strains


NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level.

Knudsen JD, Carlquist M, Gorwa-Grauslund M - AMB Express (2014)

Fluorescence plots after 24 hours of growth. (A) Histograms of the FI of the control strains carrying no XR “red dash”, wtXR “blue dash” or mutXR “green dash”). (B) Histogram of the FI of the gpd1Δ gpd2Δ strains carrying no XR (red dash), wtXR (blue dash) or mutXR (green dash). Legend TMB4140: control, no XR; TMB4141: control, wtXR; TMB4143: control, mutXR; TMB4144: gpd1Δgpd2Δ, no XR;TMB4145: gpd1Δgpd2Δ, wtXR; TMB4147:gpd1Δgpd2Δ, mutXR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230897&req=5

Figure 5: Fluorescence plots after 24 hours of growth. (A) Histograms of the FI of the control strains carrying no XR “red dash”, wtXR “blue dash” or mutXR “green dash”). (B) Histogram of the FI of the gpd1Δ gpd2Δ strains carrying no XR (red dash), wtXR (blue dash) or mutXR (green dash). Legend TMB4140: control, no XR; TMB4141: control, wtXR; TMB4143: control, mutXR; TMB4144: gpd1Δgpd2Δ, no XR;TMB4145: gpd1Δgpd2Δ, wtXR; TMB4147:gpd1Δgpd2Δ, mutXR.
Mentions: The population histogram, i.e. the amount of single cells displaying a given FI, also clearly showed differences between the strains (Figures 5A, B). After 24 hours, the different populations displayed significantly different FIs, and were thus easily distinguished in an overlay plot. This was the case both in the control (Figure 5A) and in the gpd1Δ gpd2Δ (Figure 5B) strains, with mean FIs given in channel number units of 204 (control, no XR), 160 (control, wtXR), 70 (control, mutXR) or 325 (gpd1Δ gpd2Δ, no XR), 250 (gpd1Δ gpd2Δ, wtXR) and 171 (gpd1Δ gpd2Δ, mutXR), respectively. Some overlap between strains was observed in both strain backgrounds, but it was more pronounced for the wild type strains than for the deletions strains

Bottom Line: The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in the gpd1Δgpd2Δ strain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH.In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression.The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Applied Microbiology, Department of Chemistry, Lund University, Lund, 221 00, SE, Sweden.

ABSTRACT
A reporter system was constructed to measure perturbations in the NADH/NAD(+) co-factor balance in yeast, by using the green fluorescent protein gene under the control of the GPD2 promoter that is induced under conditions of excess of NADH. High fluorescence levels were obtained in a glycerol 3-phosphate dehydrogenase double deletion strain (gpd1Δgpd2Δ), which is deficient in the ability to regenerate NAD(+) via glycerol formation. The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in the gpd1Δgpd2Δ strain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH. Addition of acetoin during cell proliferation under oxygen-limited conditions resulted in a more than 2-fold decrease in mean fluorescence intensity as compared to the control experiment. Strains carrying XRs with different selectivities for NADH could be distinguished at the single cell level, so that the XR with the highest selectivity for NADH displayed the lowest fluorescence. In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression. The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells.

No MeSH data available.


Related in: MedlinePlus