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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

Histograms of the fluorescence intensity of strains TMB4123 (gpd1Δgpd2ΔYEp-GFP, “red dash”) and TMB4144 (gpd1Δgpd2ΔYIp-GFP, “blue dash”) that were grown aerobically on glucose.
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Figure 2: Histograms of the fluorescence intensity of strains TMB4123 (gpd1Δgpd2ΔYEp-GFP, “red dash”) and TMB4144 (gpd1Δgpd2ΔYIp-GFP, “blue dash”) that were grown aerobically on glucose.

Mentions: To investigate the impact of the copy number of the reporter gene yEGFP3 and only relate the expression level to the transcription step, the reporter cassette was also transferred to an integrative vector (YIp) and introduced into the same control and gpd1Δ gpd2Δ strain backgrounds, generating strains TMB4140 (control) and TMB4144 (gpd1Δ gpd2Δ). Strains were cultivated in shake flasks with YNB media and 2% glucose and FI was recorded. Similar trend was observed as for the YEp based reporter constructs. But the FI levels were lower, with strain TMB4140 (control- YIp) only emitting 4% of the FI signal of TMB4120 (control- YEp). However, a clear difference in FI was again observed between the wild type and the double mutant, as TMB4144 (gpd1Δ gpd2Δ- YIp) displayed 21.5 fold higher FI than its corresponding control TMB 4140 (Figure 1). Also the heterogeneity of the fluorescence signal that can be measured by the standard deviation of the population was lower in the YIp system (Figure 2). Standard deviations of 44 was measured in TMB 4144 (gpd1Δ gpd2Δ- YIp) whereas a standard deviation of 172 was obtained in TMB4123 (gpd1Δ gpd2Δ- YEp). Thus, to reduce the influence of gene copy number and plasmid replication kinetics on FI output in the population, further experiments were performed using the YIp based sensor.


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

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

Histograms of the fluorescence intensity of strains TMB4123 (gpd1Δgpd2ΔYEp-GFP, “red dash”) and TMB4144 (gpd1Δgpd2ΔYIp-GFP, “blue dash”) that were grown aerobically on glucose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230897&req=5

Figure 2: Histograms of the fluorescence intensity of strains TMB4123 (gpd1Δgpd2ΔYEp-GFP, “red dash”) and TMB4144 (gpd1Δgpd2ΔYIp-GFP, “blue dash”) that were grown aerobically on glucose.
Mentions: To investigate the impact of the copy number of the reporter gene yEGFP3 and only relate the expression level to the transcription step, the reporter cassette was also transferred to an integrative vector (YIp) and introduced into the same control and gpd1Δ gpd2Δ strain backgrounds, generating strains TMB4140 (control) and TMB4144 (gpd1Δ gpd2Δ). Strains were cultivated in shake flasks with YNB media and 2% glucose and FI was recorded. Similar trend was observed as for the YEp based reporter constructs. But the FI levels were lower, with strain TMB4140 (control- YIp) only emitting 4% of the FI signal of TMB4120 (control- YEp). However, a clear difference in FI was again observed between the wild type and the double mutant, as TMB4144 (gpd1Δ gpd2Δ- YIp) displayed 21.5 fold higher FI than its corresponding control TMB 4140 (Figure 1). Also the heterogeneity of the fluorescence signal that can be measured by the standard deviation of the population was lower in the YIp system (Figure 2). Standard deviations of 44 was measured in TMB 4144 (gpd1Δ gpd2Δ- YIp) whereas a standard deviation of 172 was obtained in TMB4123 (gpd1Δ gpd2Δ- YEp). Thus, to reduce the influence of gene copy number and plasmid replication kinetics on FI output in the population, further experiments were performed using the YIp based sensor.

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