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Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria.

de Jong H, Ranquet C, Ropers D, Pinel C, Geiselmann J - BMC Syst Biol (2010)

Bottom Line: The results show that large differences in protein half-lives, more than mRNA half-lives, may be critical for the interpretation of reporter gene data in the analysis of the dynamics of regulatory systems.The paper contributes to the development of sound methods for the interpretation of reporter gene data, notably in the context of the reconstruction and validation of models of regulatory networks.The results have wide applicability for the analysis of gene expression in bacteria and may be extended to higher organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Jean Roget, LAPM, UMR5163, Campus Santé, Université Joseph Fourier, Domaine de la Merci, 38700 La Tronche, France.

ABSTRACT

Background: Fluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells. However, mathematical models are necessary for extracting biologically meaningful quantities from the primary data.

Results: We present a rigorous method for deriving relative protein synthesis rates (mRNA concentrations) and protein concentrations by means of kinetic models of gene expression. We experimentally and computationally validate this approach in the case of the protein Fis, a global regulator of transcription in Escherichia coli. We show that the mRNA and protein concentration profiles predicted from the models agree quite well with direct measurements obtained by Northern and Western blots, respectively. Moreover, we present computational procedures for taking into account systematic biases like the folding time of the fluorescent reporter protein and differences in the half-lives of reporter and host gene products. The results show that large differences in protein half-lives, more than mRNA half-lives, may be critical for the interpretation of reporter gene data in the analysis of the dynamics of regulatory systems.

Conclusions: The paper contributes to the development of sound methods for the interpretation of reporter gene data, notably in the context of the reconstruction and validation of models of regulatory networks. The results have wide applicability for the analysis of gene expression in bacteria and may be extended to higher organisms.

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Primary and corrected data. (a) Absorbance and fluorescence intensity measured on a population of bacteria carrying the GFP reporter system of Fis. (b) Absorbance and fluorescence intensity corrected for background levels. (c)-(d) Idem for the luciferase reporter system of Fis. The measurements are represented by blue circles (fluorescence or luminescence) and red crosses (absorbance), and the spline fits are indicated by solid lines. The dashed lines delimit the 95% confidence bands.
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Figure 2: Primary and corrected data. (a) Absorbance and fluorescence intensity measured on a population of bacteria carrying the GFP reporter system of Fis. (b) Absorbance and fluorescence intensity corrected for background levels. (c)-(d) Idem for the luciferase reporter system of Fis. The measurements are represented by blue circles (fluorescence or luminescence) and red crosses (absorbance), and the spline fits are indicated by solid lines. The dashed lines delimit the 95% confidence bands.

Mentions: The results obtained with the GFP and luciferase reporter plasmids of Fis are shown in Figure 2. At time zero, the growth-arrested bacterial cultures were diluted into fresh culture medium. The bacteria progressively reach the maximum growth rate in exponential phase, as can be seen with the logarithmic scale in the plots. The increase in fluorescence and luminescence levels accelerates after about one hour, but slows down later in exponential phase. When the culture enters stationary phase, the fluorescence and luminescence levels decrease due to the down-regulation of fis.


Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria.

de Jong H, Ranquet C, Ropers D, Pinel C, Geiselmann J - BMC Syst Biol (2010)

Primary and corrected data. (a) Absorbance and fluorescence intensity measured on a population of bacteria carrying the GFP reporter system of Fis. (b) Absorbance and fluorescence intensity corrected for background levels. (c)-(d) Idem for the luciferase reporter system of Fis. The measurements are represented by blue circles (fluorescence or luminescence) and red crosses (absorbance), and the spline fits are indicated by solid lines. The dashed lines delimit the 95% confidence bands.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2877006&req=5

Figure 2: Primary and corrected data. (a) Absorbance and fluorescence intensity measured on a population of bacteria carrying the GFP reporter system of Fis. (b) Absorbance and fluorescence intensity corrected for background levels. (c)-(d) Idem for the luciferase reporter system of Fis. The measurements are represented by blue circles (fluorescence or luminescence) and red crosses (absorbance), and the spline fits are indicated by solid lines. The dashed lines delimit the 95% confidence bands.
Mentions: The results obtained with the GFP and luciferase reporter plasmids of Fis are shown in Figure 2. At time zero, the growth-arrested bacterial cultures were diluted into fresh culture medium. The bacteria progressively reach the maximum growth rate in exponential phase, as can be seen with the logarithmic scale in the plots. The increase in fluorescence and luminescence levels accelerates after about one hour, but slows down later in exponential phase. When the culture enters stationary phase, the fluorescence and luminescence levels decrease due to the down-regulation of fis.

Bottom Line: The results show that large differences in protein half-lives, more than mRNA half-lives, may be critical for the interpretation of reporter gene data in the analysis of the dynamics of regulatory systems.The paper contributes to the development of sound methods for the interpretation of reporter gene data, notably in the context of the reconstruction and validation of models of regulatory networks.The results have wide applicability for the analysis of gene expression in bacteria and may be extended to higher organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Jean Roget, LAPM, UMR5163, Campus Santé, Université Joseph Fourier, Domaine de la Merci, 38700 La Tronche, France.

ABSTRACT

Background: Fluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells. However, mathematical models are necessary for extracting biologically meaningful quantities from the primary data.

Results: We present a rigorous method for deriving relative protein synthesis rates (mRNA concentrations) and protein concentrations by means of kinetic models of gene expression. We experimentally and computationally validate this approach in the case of the protein Fis, a global regulator of transcription in Escherichia coli. We show that the mRNA and protein concentration profiles predicted from the models agree quite well with direct measurements obtained by Northern and Western blots, respectively. Moreover, we present computational procedures for taking into account systematic biases like the folding time of the fluorescent reporter protein and differences in the half-lives of reporter and host gene products. The results show that large differences in protein half-lives, more than mRNA half-lives, may be critical for the interpretation of reporter gene data in the analysis of the dynamics of regulatory systems.

Conclusions: The paper contributes to the development of sound methods for the interpretation of reporter gene data, notably in the context of the reconstruction and validation of models of regulatory networks. The results have wide applicability for the analysis of gene expression in bacteria and may be extended to higher organisms.

Show MeSH
Related in: MedlinePlus