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Stochastic protein expression in individual cells at the single molecule level

Abstract

In a living cell, gene expression—the transcription of DNA to messenger RNA followed by translation to protein—occurs stochastically, as a consequence of the low copy number of DNA and mRNA molecules involved1,2,3,4,5,6. These stochastic events of protein production are difficult to observe directly with measurements on large ensembles of cells owing to lack of synchronization among cells. Measurements so far on single cells lack the sensitivity to resolve individual events of protein production. Here we demonstrate a microfluidic-based assay that allows real-time observation of the expression of β-galactosidase in living Escherichia coli cells with single molecule sensitivity. We observe that protein production occurs in bursts, with the number of molecules per burst following an exponential distribution. We show that the two key parameters of protein expression—the burst size and frequency—can be either determined directly from real-time monitoring of protein production or extracted from a measurement of the steady-state copy number distribution in a population of cells. Application of this assay to probe gene expression in individual budding yeast and mouse embryonic stem cells demonstrates its generality. Many important proteins are expressed at low levels7,8, and are thus inaccessible by current genomic and proteomic techniques. This microfluidic single cell assay opens up possibilities for system-wide characterization of the expression of these low copy number proteins.

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Figure 1: Single reporter molecule sensitivity in a microfluidic device.
Figure 2: Quantitative real-time measurement of individual protein expression events in live E. coli cells.
Figure 3: Steady-state protein copy number distributions in a population of cells.
Figure 4: Two regimes of stochasticity in protein expression.

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Acknowledgements

We acknowledge J. Xiao, K. Gudiksen and J. Markson for early involvement in the project, and J. Yu, P. Choi and J. Elf for discussions and careful reading of the manuscript. We are grateful to Q. Xu, H. Wu, members of G. M. Whitesides and R. N. Zare groups, and the Harvard Center for Nanoscale Systems for help with microfluidic fabrication. We thank K. Thorn for yeast strains, and A. and J. McMahon for stem cell strains. This work was funded by Department of Energy, Office of Biological and Environmental Research, Genomics: GTL Program, and in part by National Institute of Health (NIH) Director's Pioneer Award to X.S.X., and an NIH grant. L.C. is supported by a National Science Foundation Graduate Research Fellowship and N.F. by a Human Frontiers Science Program Long Term Fellowship.

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Correspondence to X. Sunney Xie.

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This file contains the Supplementary Methods, Supplementary Figures 1–12 and additional references. (DOC 280 kb)

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Cai, L., Friedman, N. & Xie, X. Stochastic protein expression in individual cells at the single molecule level. Nature 440, 358–362 (2006). https://doi.org/10.1038/nature04599

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