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Tunable translational control using site-specific unnatural amino acid incorporation in Escherichia coli.

Kato Y - PeerJ (2015)

Bottom Line: Here, we report that this translational switch can control the translational efficiency at any intermediate magnitude by adjustment of the 3-iodo-L-tyrosine concentration in the medium, as a tunable translational controller.The translational efficiency of a target gene reached maximum levels with 10(-5) M 3-iodo-L-tyrosine, and intermediate levels were observed with suboptimal concentrations (approximately spanning a 2-log10 concentration range, 10(-7)-10(-5) M).Such intermediate-level expression was also confirmed in individual bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences , Tsukuba, Ibaraki , Japan.

ABSTRACT
Translation of target gene transcripts in Escherichia coli harboring UAG amber stop codons can be switched on by the amber-codon-specific incorporation of an exogenously supplied unnatural amino acid, 3-iodo-L-tyrosine. Here, we report that this translational switch can control the translational efficiency at any intermediate magnitude by adjustment of the 3-iodo-L-tyrosine concentration in the medium, as a tunable translational controller. The translational efficiency of a target gene reached maximum levels with 10(-5) M 3-iodo-L-tyrosine, and intermediate levels were observed with suboptimal concentrations (approximately spanning a 2-log10 concentration range, 10(-7)-10(-5) M). Such intermediate-level expression was also confirmed in individual bacteria.

No MeSH data available.


Related in: MedlinePlus

EGFP expression in individual bacteria.The sampling points are indicated in the dose-response curve in Fig. 2A. Upper and lower photographs are epifluorescence images and Nomarski differential interference contrast images, respectively. The photographic conditions were constant for all of the fluorescence images. Calibration bar = 100 µm. Asterisks in spatial distribution graphs indicate the upper right corners of the fluorescence images. The frequency in the histograms indicates the number of individual bacteria.
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fig-3: EGFP expression in individual bacteria.The sampling points are indicated in the dose-response curve in Fig. 2A. Upper and lower photographs are epifluorescence images and Nomarski differential interference contrast images, respectively. The photographic conditions were constant for all of the fluorescence images. Calibration bar = 100 µm. Asterisks in spatial distribution graphs indicate the upper right corners of the fluorescence images. The frequency in the histograms indicates the number of individual bacteria.

Mentions: The experimental system is schematically shown in Fig. 1 (A) Dose-response curve. Bacteria were cultured in media containing various concentrations of IY for 6 h. Colored circles indicate the samples for Fig. 3. Data are shown as mean ± SEM. n = 3 independent experiments using completely separated bacterial cultures. Statistical analysis was performed using Welch’s t-test in Excel ver. 14.0. A single fitted dose-response curve (log-logistic, IY = 0–3 × 10−4 M) was generated using Origin7. The equation for the fitted curve and assessments of goodness-of-fit are represented under the graph. F, fluorescent intensity (arbitrary unit); Fmax, the initial F value (right horizontal asymptote); Fmin, the final F value (left horizontal asymptote); CIY, IY concentration (M); C0, 50% effective CIY (point of inflection); χ2, reduced chi-squared value; DoF, degrees of freedom. (B) Time course of EGFP accumulation.


Tunable translational control using site-specific unnatural amino acid incorporation in Escherichia coli.

Kato Y - PeerJ (2015)

EGFP expression in individual bacteria.The sampling points are indicated in the dose-response curve in Fig. 2A. Upper and lower photographs are epifluorescence images and Nomarski differential interference contrast images, respectively. The photographic conditions were constant for all of the fluorescence images. Calibration bar = 100 µm. Asterisks in spatial distribution graphs indicate the upper right corners of the fluorescence images. The frequency in the histograms indicates the number of individual bacteria.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4419535&req=5

fig-3: EGFP expression in individual bacteria.The sampling points are indicated in the dose-response curve in Fig. 2A. Upper and lower photographs are epifluorescence images and Nomarski differential interference contrast images, respectively. The photographic conditions were constant for all of the fluorescence images. Calibration bar = 100 µm. Asterisks in spatial distribution graphs indicate the upper right corners of the fluorescence images. The frequency in the histograms indicates the number of individual bacteria.
Mentions: The experimental system is schematically shown in Fig. 1 (A) Dose-response curve. Bacteria were cultured in media containing various concentrations of IY for 6 h. Colored circles indicate the samples for Fig. 3. Data are shown as mean ± SEM. n = 3 independent experiments using completely separated bacterial cultures. Statistical analysis was performed using Welch’s t-test in Excel ver. 14.0. A single fitted dose-response curve (log-logistic, IY = 0–3 × 10−4 M) was generated using Origin7. The equation for the fitted curve and assessments of goodness-of-fit are represented under the graph. F, fluorescent intensity (arbitrary unit); Fmax, the initial F value (right horizontal asymptote); Fmin, the final F value (left horizontal asymptote); CIY, IY concentration (M); C0, 50% effective CIY (point of inflection); χ2, reduced chi-squared value; DoF, degrees of freedom. (B) Time course of EGFP accumulation.

Bottom Line: Here, we report that this translational switch can control the translational efficiency at any intermediate magnitude by adjustment of the 3-iodo-L-tyrosine concentration in the medium, as a tunable translational controller.The translational efficiency of a target gene reached maximum levels with 10(-5) M 3-iodo-L-tyrosine, and intermediate levels were observed with suboptimal concentrations (approximately spanning a 2-log10 concentration range, 10(-7)-10(-5) M).Such intermediate-level expression was also confirmed in individual bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences , Tsukuba, Ibaraki , Japan.

ABSTRACT
Translation of target gene transcripts in Escherichia coli harboring UAG amber stop codons can be switched on by the amber-codon-specific incorporation of an exogenously supplied unnatural amino acid, 3-iodo-L-tyrosine. Here, we report that this translational switch can control the translational efficiency at any intermediate magnitude by adjustment of the 3-iodo-L-tyrosine concentration in the medium, as a tunable translational controller. The translational efficiency of a target gene reached maximum levels with 10(-5) M 3-iodo-L-tyrosine, and intermediate levels were observed with suboptimal concentrations (approximately spanning a 2-log10 concentration range, 10(-7)-10(-5) M). Such intermediate-level expression was also confirmed in individual bacteria.

No MeSH data available.


Related in: MedlinePlus