Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals

Nature Methods volume 11, pages 449–455 (2014)Cite this article

Subjects

Abstract

Gene circuits are dynamical systems that regulate cellular behaviors, often using protein signals as inputs and outputs. Here we have developed an optogenetic 'function generator' method for programming tailor-made gene expression signals in live bacterial cells. We designed precomputed light sequences based on experimentally calibrated mathematical models of light-switchable two-component systems and used them to drive intracellular protein levels to match user-defined reference time courses. We used this approach to generate accelerated and linearized dynamics, sinusoidal oscillations with desired amplitudes and periods, and a complex waveform, all with unprecedented accuracy and precision. We also combined the function generator with a dual fluorescent protein reporter system, analogous to a dual-channel oscilloscope, to reveal that a synthetic repressible promoter linearly transforms repressor signals with an approximate 7-min delay. Our approach will enable a new generation of dynamical analyses of synthetic and natural gene circuits, providing an essential step toward the predictive design and rigorous understanding of biological systems.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Light-switchable two-component systems (TCSs) and light tube array (LTA).
Figure 2: Experimentally characterized TCS models predict input-output dynamics.
Figure 3: Programming protein transition dynamics with a biological function generator.
Figure 4: Programming custom protein-expression signals with a biological function generator.
Figure 5: Analysis of gene circuit signal processing using the biological function generator and dual-reporter oscilloscope system.

References

  1. Bennett, M.R. et al. Metabolic gene regulation in a dynamically changing environment. Nature 454, 1119–1122 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Mettetal, J.T., Muzzey, D., Gómez-Uribe, C. & van Oudenaarden, A. The frequency dependence of osmo-adaptation in Saccharomyces cerevisiae. Science 319, 482–484 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wei, P. et al. Bacterial virulence proteins as tools to rewire kinase pathways in yeast and immune cells. Nature 488, 384–388 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Muzzey, D., Gómez-Uribe, C.A., Mettetal, J.T. & van Oudenaarden, A. A systems-level analysis of perfect adaptation in yeast osmoregulation. Cell 138, 160–171 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tan, C. et al. The inoculum effect and band-pass bacterial response to periodic antibiotic treatment. Mol. Syst. Biol. 8, 617 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Hersen, P., McClean, M.N., Mahadevan, L. & Ramanathan, S. Signal processing by the HOG MAP kinase pathway. Proc. Natl. Acad. Sci. USA 105, 7165–7170 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Milias-Argeitis, A. et al. In silico feedback for in vivo regulation of a gene expression circuit. Nat. Biotechnol. 29, 1114–1116 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Uhlendorf, J. et al. Long-term model predictive control of gene expression at the population and single-cell levels. Proc. Natl. Acad. Sci. USA 109, 14271–14276 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tabor, J.J., Levskaya, A. & Voigt, C.A. Multichromatic control of gene expression in Escherichia coli. J. Mol. Biol. 405, 315–324 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Levskaya, A. et al. Synthetic biology: engineering Escherichia coli to see light. Nature 438, 441–442 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1–I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T.C. & Waldo, G.S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

    Article  PubMed  Google Scholar 

  13. Alon, U. An Introduction to Systems Biology: Design Principles of Biological Networks (CRC Press, 2007).

  14. Yokobayashi, Y., Weiss, R. & Arnold, F.H. Directed evolution of a genetic circuit. Proc. Natl. Acad. Sci. USA 99, 16587–16591 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ang, J., Ingalls, B. & McMillen, D. Probing the input-output behavior of biochemical and genetic systems system identification methods from control theory. Methods Enzymol. 487, 279–317 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Levine, J.H., Lin, Y. & Elowitz, M.B. Functional roles of pulsing in genetic circuits. Science 342, 1193–1200 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. McAdams, H.H. & Shapiro, L. A bacterial cell-cycle regulatory network operating in time and space. Science 301, 1874–1877 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Bell-Pedersen, D. et al. Circadian rhythms from multiple oscillators: lessons from diverse organisms. Nat. Rev. Genet. 6, 544–556 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Young, J.W., Locke, J.C.W. & Elowitz, M.B. Rate of environmental change determines stress response specificity. Proc. Natl. Acad. Sci. USA 110, 4140–4145 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. El-Samad, H., Kurata, H., Doyle, J.C., Gross, C.A. & Khammash, M. Surviving heat shock: control strategies for robustness and performance. Proc. Natl. Acad. Sci. USA 102, 2736–2741 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Vishnoi, M. et al. Triggering sporulation in Bacillus subtilis with artificial two-component systems reveals the importance of proper Spo0A activation dynamics. Mol. Microbiol. 90, 181–194 (2013).

    CAS  PubMed  Google Scholar 

  22. Levine, J.H., Fontes, M.E., Dworkin, J. & Elowitz, M.B. Pulsed feedback defers cellular differentiation. PLoS Biol. 10, e1001252 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kuchina, A. et al. Temporal competition between differentiation programs determines cell fate choice. Mol. Syst. Biol. 7, 557 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Ray, J.C.J., Tabor, J.J. & Igoshin, O.A. Non-transcriptional regulatory processes shape transcriptional network dynamics. Nat. Rev. Microbiol. 9, 817–828 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kalir, S. et al. Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science 292, 2080–2083 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Temme, K. et al. Induction and relaxation dynamics of the regulatory network controlling the type III secretion system encoded within Salmonella pathogenicity island 1. J. Mol. Biol. 377, 47–61 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Cardinale, S., Joachimiak, M.P. & Arkin, A.P. Effects of genetic variation on the E. coli host-circuit interface. Cell Rep. 4, 231–237 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Arkin, A.P. A wise consistency: engineering biology for conformity, reliability, predictability. Curr. Opin. Chem. Biol. 17, 893–901 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat. Biotechnol. 26, 787–793 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Zaslaver, A. et al. Just-in-time transcription program in metabolic pathways. Nat. Genet. 36, 486–491 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Temme, K., Zhao, D. & Voigt, C.A. Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc. Natl. Acad. Sci. USA 109, 7085–7090 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Rockwell, N.C., Martin, S.S., Feoktistova, K. & Lagarias, J.C. Diverse two-cysteine photocycles in phytochromes and cyanobacteriochromes. Proc. Natl. Acad. Sci. USA 108, 11854–11859 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jaubert, M. et al. Control of peripheral light-harvesting complex synthesis by a bacteriophytochrome in the aerobic photosynthetic bacterium Bradyrhizobium strain BTAi1. J. Bacteriol. 190, 5824–5831 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Shimizu-Sato, S., Huq, E., Tepperman, J.M. & Quail, P.H. A light-switchable gene promoter system. Nat. Biotechnol. 20, 1041–1044 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Müller, K. et al. Multi-chromatic control of mammalian gene expression and signaling. Nucleic Acids Res. 41, e124 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Levskaya, A., Weiner, O.D., Lim, W.A. & Voigt, C.A. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Toettcher, J.E., Gong, D., Lim, W.A. & Weiner, O.D. Light-based feedback for controlling intracellular signaling dynamics. Nat. Methods 8, 837–839 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Toettcher, J.E., Weiner, O.D. & Lim, W.A. Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155, 1422–1434 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Miyazaki, K. MEGAWHOP cloning: a method of creating random mutagenesis libraries via megaprimer PCR of whole plasmids. Methods Enzymol. 498, 399–406 (2011).

    Article  CAS  PubMed  Google Scholar 

  40. Salis, H.M., Mirsky, E.A. & Voigt, C.A. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946–950 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    CAS  PubMed  Google Scholar 

  42. Tabor, J.J. et al. A synthetic genetic edge detection program. Cell 137, 1272–1281 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3, e3647 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by the US National Science Foundation Biotechnology, Biochemical, and Biomass Engineering (BBBE) program (EFRI–1137266) and the Office of Naval Research MURI programs (N000141310074). L.A.H. was supported by the US National Aeronautics and Space Administration Office of the Chief Technologist's Space Technology Research Fellowship (NSTRF) (NNX11AN39H). We thank S. Schmidl (Rice University) for his contribution of the pPCBSE plasmid.

Author information

Authors and Affiliations

  1. Graduate Program in Applied Physics, Rice University, Houston, Texas, USA

    Evan J Olson

  2. Department of Bioengineering, Rice University, Houston, Texas, USA

    Lucas A Hartsough, Brian P Landry, Raghav Shroff & Jeffrey J Tabor

  3. Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, USA

    Jeffrey J Tabor

Authors
  1. Evan J Olson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lucas A Hartsough
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Brian P Landry
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Raghav Shroff
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey J Tabor
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.J.T. and E.J.O. conceived of the project, and J.J.T. supervised the project. E.J.O., L.A.H., B.P.L. and R.S. designed and performed experiments. E.J.O. and B.P.L. designed and constructed plasmids. E.J.O. designed and constructed the LTA, analyzed data, constructed the model and developed the light program sequence optimization algorithm. J.J.T. and E.J.O. wrote the manuscript.

Corresponding author

Correspondence to Jeffrey J Tabor.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18, Supplementary Tables 1–7 and Supplementary Notes 1–12 (PDF 3922 kb)

Supplementary Software

Python scripts to perform flow cytometry file extraction and analysis, produce model simulations of gene expression output, fit models to gene expression data and compute light inputs that drive gene expression output to follow a reference signal. (ZIP 1580 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Olson, E., Hartsough, L., Landry, B. et al. Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals. Nat Methods 11, 449–455 (2014). https://doi.org/10.1038/nmeth.2884

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2884

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing