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Single-cell zeroth-order protein degradation enhances the robustness of synthetic oscillator.

Wong WW, Tsai TY, Liao JC - Mol. Syst. Biol. (2007)

Bottom Line: In Escherichia coli, protein degradation in synthetic circuits is commonly achieved by the ssrA-tagged degradation system.In this work, we show that the degradation kinetics for the green fluorescent protein fused with the native ssrA tag in each cell exhibits the zeroth-order limit of the Michaelis-Menten kinetics, rather than the commonly assumed first-order.When measured in a population, the wide distribution of protein levels in the cells distorts the true kinetics and results in a first-order protein degradation kinetics as a population average.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, California 90095, USA.

ABSTRACT
In Escherichia coli, protein degradation in synthetic circuits is commonly achieved by the ssrA-tagged degradation system. In this work, we show that the degradation kinetics for the green fluorescent protein fused with the native ssrA tag in each cell exhibits the zeroth-order limit of the Michaelis-Menten kinetics, rather than the commonly assumed first-order. When measured in a population, the wide distribution of protein levels in the cells distorts the true kinetics and results in a first-order protein degradation kinetics as a population average. Using the synthetic gene-metabolic oscillator constructed previously, we demonstrated theoretically that the zeroth-order kinetics significantly enlarges the parameter space for oscillation and thus enhances the robustness of the design under parametric uncertainty.

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Effect of zero-order degradation kinetics on the metabolator. (A) Network diagrams of the metabolator. The promoter glnAp2 is activated by AcP. The promoter lacO1 is repressed by LacI. All proteins are tagged with the LAA degradation tag (Fung et al, 2005). (B) Phase diagram of the zeroth-order degradation rate, kd,0, and first-order degradation rate, kd,1, for the metabolator model. The presence of zeroth-order protein degradation enlarges the parameter space for oscillation. When the zero-order degradation rate is zero (x-axis), the current model reduces to the original model. The point on the x-axis indicates the degradation rate used in the original metabolator model (see Supplementary Information for details of the model and parameters). (C) Phase diagram of the relative copy numbers acs and pta to lacI when the degradation kinetics is zeroth- and first-order. (D) Oscillation dynamics using parameters that lay outside of the first-order region, but inside the zero-order region. The point is represented by a triangle in (C).
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f4: Effect of zero-order degradation kinetics on the metabolator. (A) Network diagrams of the metabolator. The promoter glnAp2 is activated by AcP. The promoter lacO1 is repressed by LacI. All proteins are tagged with the LAA degradation tag (Fung et al, 2005). (B) Phase diagram of the zeroth-order degradation rate, kd,0, and first-order degradation rate, kd,1, for the metabolator model. The presence of zeroth-order protein degradation enlarges the parameter space for oscillation. When the zero-order degradation rate is zero (x-axis), the current model reduces to the original model. The point on the x-axis indicates the degradation rate used in the original metabolator model (see Supplementary Information for details of the model and parameters). (C) Phase diagram of the relative copy numbers acs and pta to lacI when the degradation kinetics is zeroth- and first-order. (D) Oscillation dynamics using parameters that lay outside of the first-order region, but inside the zero-order region. The point is represented by a triangle in (C).

Mentions: We also investigated the effect of protein degradation on synthetic oscillators. We used the metabolator (Fung et al, 2005) as an example, which is a synthetic gene-metabolic oscillator that integrates transcriptional regulation into the metabolism to generate oscillation. The metabolator is consisted of a flux-carrying network with two interconvertible metabolite pools: Acetyl-CoA (AcCoA) and Acetyl-phosphate (AcP) (Figure 4A). These two pools of metabolites are catalyzed by two enzymes, phosphotransacetylase (pta) and acetyl-CoA synthetase (acs). The expression of pta and acs are negatively and positively regulated by AcP, respectively. The oscillation dynamics of the metabolator is driven by the glycolytic flux. The integration of genetic and metabolic control is a hallmark found in many natural oscillators (Hirota et al, 2002; Rutter et al, 2002; Rudic et al, 2004; Turek et al, 2005).


Single-cell zeroth-order protein degradation enhances the robustness of synthetic oscillator.

Wong WW, Tsai TY, Liao JC - Mol. Syst. Biol. (2007)

Effect of zero-order degradation kinetics on the metabolator. (A) Network diagrams of the metabolator. The promoter glnAp2 is activated by AcP. The promoter lacO1 is repressed by LacI. All proteins are tagged with the LAA degradation tag (Fung et al, 2005). (B) Phase diagram of the zeroth-order degradation rate, kd,0, and first-order degradation rate, kd,1, for the metabolator model. The presence of zeroth-order protein degradation enlarges the parameter space for oscillation. When the zero-order degradation rate is zero (x-axis), the current model reduces to the original model. The point on the x-axis indicates the degradation rate used in the original metabolator model (see Supplementary Information for details of the model and parameters). (C) Phase diagram of the relative copy numbers acs and pta to lacI when the degradation kinetics is zeroth- and first-order. (D) Oscillation dynamics using parameters that lay outside of the first-order region, but inside the zero-order region. The point is represented by a triangle in (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1943427&req=5

f4: Effect of zero-order degradation kinetics on the metabolator. (A) Network diagrams of the metabolator. The promoter glnAp2 is activated by AcP. The promoter lacO1 is repressed by LacI. All proteins are tagged with the LAA degradation tag (Fung et al, 2005). (B) Phase diagram of the zeroth-order degradation rate, kd,0, and first-order degradation rate, kd,1, for the metabolator model. The presence of zeroth-order protein degradation enlarges the parameter space for oscillation. When the zero-order degradation rate is zero (x-axis), the current model reduces to the original model. The point on the x-axis indicates the degradation rate used in the original metabolator model (see Supplementary Information for details of the model and parameters). (C) Phase diagram of the relative copy numbers acs and pta to lacI when the degradation kinetics is zeroth- and first-order. (D) Oscillation dynamics using parameters that lay outside of the first-order region, but inside the zero-order region. The point is represented by a triangle in (C).
Mentions: We also investigated the effect of protein degradation on synthetic oscillators. We used the metabolator (Fung et al, 2005) as an example, which is a synthetic gene-metabolic oscillator that integrates transcriptional regulation into the metabolism to generate oscillation. The metabolator is consisted of a flux-carrying network with two interconvertible metabolite pools: Acetyl-CoA (AcCoA) and Acetyl-phosphate (AcP) (Figure 4A). These two pools of metabolites are catalyzed by two enzymes, phosphotransacetylase (pta) and acetyl-CoA synthetase (acs). The expression of pta and acs are negatively and positively regulated by AcP, respectively. The oscillation dynamics of the metabolator is driven by the glycolytic flux. The integration of genetic and metabolic control is a hallmark found in many natural oscillators (Hirota et al, 2002; Rutter et al, 2002; Rudic et al, 2004; Turek et al, 2005).

Bottom Line: In Escherichia coli, protein degradation in synthetic circuits is commonly achieved by the ssrA-tagged degradation system.In this work, we show that the degradation kinetics for the green fluorescent protein fused with the native ssrA tag in each cell exhibits the zeroth-order limit of the Michaelis-Menten kinetics, rather than the commonly assumed first-order.When measured in a population, the wide distribution of protein levels in the cells distorts the true kinetics and results in a first-order protein degradation kinetics as a population average.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, California 90095, USA.

ABSTRACT
In Escherichia coli, protein degradation in synthetic circuits is commonly achieved by the ssrA-tagged degradation system. In this work, we show that the degradation kinetics for the green fluorescent protein fused with the native ssrA tag in each cell exhibits the zeroth-order limit of the Michaelis-Menten kinetics, rather than the commonly assumed first-order. When measured in a population, the wide distribution of protein levels in the cells distorts the true kinetics and results in a first-order protein degradation kinetics as a population average. Using the synthetic gene-metabolic oscillator constructed previously, we demonstrated theoretically that the zeroth-order kinetics significantly enlarges the parameter space for oscillation and thus enhances the robustness of the design under parametric uncertainty.

Show MeSH
Related in: MedlinePlus