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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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Related in: MedlinePlus

Correction of GFP concentration and synthesis rate for folding time. (a) Concentration profile of active GFP (blue line) and total GFP (green line). The latter profile has been corrected for the folding time of GFP. Both profiles are normalized with respect to the peak in mid-exponential phase of the active GFP concentration. (b) Concentration profile of gfp mRNA, before correction (blue line) and after correction (green line) for the folding time. Normalization is as in panel a.
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Figure 8: Correction of GFP concentration and synthesis rate for folding time. (a) Concentration profile of active GFP (blue line) and total GFP (green line). The latter profile has been corrected for the folding time of GFP. Both profiles are normalized with respect to the peak in mid-exponential phase of the active GFP concentration. (b) Concentration profile of gfp mRNA, before correction (blue line) and after correction (green line) for the folding time. Normalization is as in panel a.

Mentions: Figure 8a shows the concentration profiles of both active and total GFP, normalized with respect to the peak in mid-exponential phase of the active GFP concentration. As expected, active GFP represents only a fraction of total GFP. However, the qualitative shape of the profiles remains essentially the same. Using the profile of q(t) instead of r(t) for computing the normalized reporter synthesis rate, and thus the normalized mRNA concentration, yields the same conclusion (Figure 8b). In both cases we see that the expression peak is slightly shifted to an earlier time-point. This is consistent with the fact that the maturation process introduces a delay in the availability of active GFP. The agreement of the computed profiles with the Western and Northern blot measurements is not improved by correcting for the folding time (result not shown).


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)

Correction of GFP concentration and synthesis rate for folding time. (a) Concentration profile of active GFP (blue line) and total GFP (green line). The latter profile has been corrected for the folding time of GFP. Both profiles are normalized with respect to the peak in mid-exponential phase of the active GFP concentration. (b) Concentration profile of gfp mRNA, before correction (blue line) and after correction (green line) for the folding time. Normalization is as in panel a.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Correction of GFP concentration and synthesis rate for folding time. (a) Concentration profile of active GFP (blue line) and total GFP (green line). The latter profile has been corrected for the folding time of GFP. Both profiles are normalized with respect to the peak in mid-exponential phase of the active GFP concentration. (b) Concentration profile of gfp mRNA, before correction (blue line) and after correction (green line) for the folding time. Normalization is as in panel a.
Mentions: Figure 8a shows the concentration profiles of both active and total GFP, normalized with respect to the peak in mid-exponential phase of the active GFP concentration. As expected, active GFP represents only a fraction of total GFP. However, the qualitative shape of the profiles remains essentially the same. Using the profile of q(t) instead of r(t) for computing the normalized reporter synthesis rate, and thus the normalized mRNA concentration, yields the same conclusion (Figure 8b). In both cases we see that the expression peak is slightly shifted to an earlier time-point. This is consistent with the fact that the maturation process introduces a delay in the availability of active GFP. The agreement of the computed profiles with the Western and Northern blot measurements is not improved by correcting for the folding time (result not shown).

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