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Comparison and Calibration of Different Reporters for Quantitative Analysis of Gene Expression

Garcia, Hernan G. and Lee, Heun Jin and Boedicker, James Q. and Phillips, Rob (2011) Comparison and Calibration of Different Reporters for Quantitative Analysis of Gene Expression. Biophysical Journal, 101 (3). pp. 535-544. ISSN 0006-3495. https://resolver.caltech.edu/CaltechAUTHORS:20110929-140259410

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Abstract

Absolute levels of gene expression in bacteria are observed to vary over as much as six orders of magnitude. Thermodynamic models have been proposed as a tool to describe the expression levels of a given transcriptional circuit. In this context, it is essential to understand both the limitations and linear range of the different methods for measuring gene expression and to determine to what extent measurements from different reporters can be directly compared with one aim being the stringent testing of theoretical descriptions of gene expression. In this article, we compare two protein reporters by measuring both the absolute level of expression and fold-change in expression using the fluorescent protein EYFP and the enzymatic reporter β-galactosidase. We determine their dynamic and linear range and show that they are interchangeable for measuring mean levels of expression over four orders of magnitude. By calibrating these reporters such that they can be interpreted in terms of absolute molecular counts, we establish limits for their applicability: autofluorescence on the lower end of expression for EYFP (at ∼10 molecules per cell) and interference with cellular growth on the high end for β-galactosidase (at ∼20,000 molecules per cell). These qualities make the reporters complementary and necessary when trying to experimentally verify the predictions from the theoretical models.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.bpj.2011.06.026DOIUNSPECIFIED
http://www.sciencedirect.com/science/article/pii/S0006349511007557PublisherUNSPECIFIED
Additional Information:© 2011 by the Biophysical Society. Received 17 November 2010; Accepted 9 June 2011. Editor: Andre Levchenko. Available online 2 August 2011. We are grateful to a number of people for instruction, helpful discussions, and reading the manuscript. In particular, we are grateful to Nicole Becker, Jim Maher, Bob Schleif, Terry Hwa, James Locke, Ron Milo, Tom Kuhlman, Alvaro Sanchez, and Franz Weinert. We thank Roberto Chica for help with the EYFP purification. This work was supported by National Institutes of Health Pioneer award No. DP1 OD000217 (to H.G.G., H.J.L., and R.P.), National Institutes of Health award No. R01 GM085286 and No. R01 GM085286-01S (to H.G.G., J.Q.B., and R.P.), La Fondation Pierre Gilles de Gennes (to R.P.), and the Caltech Division of Biology (to H.J.L.).
Funders:
Funding AgencyGrant Number
NIH Directors Pioneer Award DP1 OD000217
NIH R01 GM085286
NIHR01 GM085286-01S
La Fondation Pierre Gilles de Gennes UNSPECIFIED
Caltech Division of BiologyUNSPECIFIED
Issue or Number:3
Record Number:CaltechAUTHORS:20110929-140259410
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20110929-140259410
Official Citation:Hernan G. Garcia, Heun Jin Lee, James Q. Boedicker, Rob Phillips, Comparison and Calibration of Different Reporters for Quantitative Analysis of Gene Expression, Biophysical Journal, Volume 101, Issue 3, 3 August 2011, Pages 535-544, ISSN 0006-3495, 10.1016/j.bpj.2011.06.026. (http://www.sciencedirect.com/science/article/pii/S0006349511007557)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:25499
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:30 Sep 2011 15:20
Last Modified:03 Oct 2019 03:06

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