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.

  • Article
  • Published:

Digital switching in a biosensor circuit via programmable timing of gene availability

Nature Chemical Biology volume 10, pages 1020–1027 (2014)Cite this article

Subjects

Abstract

Transient delivery of gene circuits is required in many potential applications of synthetic biology, yet the pre-steady-state processes that dominate this delivery route pose major challenges for robust circuit deployment. Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multigene circuits. We exemplify the concept with a proportional sensor for endogenous microRNA (miRNA) and show a marked reduction in its ground state leakage due to desynchronization of the circuit's repressor components and their repression target. The new sensors display a dynamic range of up to 1,000-fold compared to 20-fold in the standard configuration. We applied the approach to classify cell types on the basis of miRNA expression profile and measured >200-fold output differential between positively and negatively identified cells. We also showed major improvements in specificity with cytotoxic output. Our study opens new venues in gene circuit design via judicious temporal control of circuits' genetic makeup.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Source of leakage in the proportional sensor.
Figure 2: The effect of delayed output on sensor performance.
Figure 3: Control of the timing.
Figure 4: Component contribution to sensor performance.
Figure 5: Cell type classification.
Figure 6: Selective cytotoxicity in HEK293 and HCT-116 cells with HSV-TK output.

Similar content being viewed by others

References

  1. Wodarz, A. & Nusse, R. Mechanisms of Wnt signaling in development. Annu. Rev. Cell Dev. Biol. 14, 59–88 (1998).

    Article  CAS  Google Scholar 

  2. Shimizu, T.S., Tu, Y.H. & Berg, H.C. A modular gradient-sensing network for chemotaxis in Escherichia coli revealed by responses to time-varying stimuli. Mol. Syst. Biol. 6, 382 (2010).

    Article  Google Scholar 

  3. Mansfield, J.H. et al. MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat. Genet. 36, 1079–1083 (2004).

    Article  CAS  Google Scholar 

  4. Zhang, F., Carothers, J.M. & Keasling, J.D. Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat. Biotechnol. 30, 354–359 (2012).

    Article  CAS  Google Scholar 

  5. Weber, W. et al. A synthetic mammalian gene circuit reveals antituberculosis compounds. Proc. Natl. Acad. Sci. USA 105, 9994–9998 (2008).

    Article  CAS  Google Scholar 

  6. Callura, J.M., Dwyer, D.J., Isaacs, F.J., Cantor, C.R. & Collins, J.J. Tracking, tuning and terminating microbial physiology using synthetic riboregulators. Proc. Natl. Acad. Sci. USA 107, 15898–15903 (2010).

    Article  CAS  Google Scholar 

  7. Gardner, T.S., Cantor, C.R. & Collins, J.J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).

    Article  CAS  Google Scholar 

  8. Egbert, R.G. & Klavins, E. Fine-tuning gene networks using simple sequence repeats. Proc. Natl. Acad. Sci. USA 109, 16817–16822 (2012).

    Article  CAS  Google Scholar 

  9. Basu, S., Gerchman, Y., Collins, C.H., Arnold, F.H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature 434, 1130–1134 (2005).

    Article  CAS  Google Scholar 

  10. de Las Heras, A., Carreno, C.A., Martinez-Garcia, E. & de Lorenzo, V. Engineering input/output nodes in prokaryotic regulatory circuits. FEMS Microbiol. Rev. 34, 842–865 (2010).

    Article  CAS  Google Scholar 

  11. Liang, J.C., Chang, A.L., Kennedy, A.B. & Smolke, C.D. A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity. Nucleic Acids Res. 40, e154 (2012).

    Article  CAS  Google Scholar 

  12. Weber, W. et al. A synthetic time-delay circuit in mammalian cells and mice. Proc. Natl. Acad. Sci. USA 104, 2643–2648 (2007).

    Article  CAS  Google Scholar 

  13. Elowitz, M.B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).

    Article  CAS  Google Scholar 

  14. Prindle, A. et al. A sensing array of radically coupled genetic 'biopixels'. Nature 481, 39–44 (2012).

    Article  CAS  Google Scholar 

  15. 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  Google Scholar 

  16. Rinaudo, K. et al. A universal RNAi-based logic evaluator that operates in mammalian cells. Nat. Biotechnol. 25, 795–801 (2007).

    Article  CAS  Google Scholar 

  17. Deans, T.L., Cantor, C.R. & Collins, J.J. A tunable genetic switch based on RNAi and repressor proteins for regulating gene expression in mammalian cells. Cell 130, 363–372 (2007).

    Article  CAS  Google Scholar 

  18. Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R. & Benenson, Y. Multi-input RNAi-based logic circuit for identification of specific cancer cells. Science 333, 1307–1311 (2011).

    Article  CAS  Google Scholar 

  19. Haynes, K.A., Ceroni, F., Flicker, D., Younger, A. & Silver, P.A. A sensitive switch for visualizing natural gene silencing in single cells. ACS Synth. Biol. 1, 99–106 (2012).

    Article  CAS  Google Scholar 

  20. Biffi, A. et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 341, 1233158 (2013).

    Article  Google Scholar 

  21. Kota, J. et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 137, 1005–1017 (2009).

    Article  CAS  Google Scholar 

  22. Mangan, S. & Alon, U. Structure and function of the feed-forward loop network motif. Proc. Natl. Acad. Sci. USA 100, 11980–11985 (2003).

    Article  CAS  Google Scholar 

  23. Basu, S., Mehreja, R., Thiberge, S., Chen, M.T. & Weiss, R. Spatiotemporal control of gene expression with pulse-generating networks. Proc. Natl. Acad. Sci. USA 101, 6355–6360 (2004).

    Article  CAS  Google Scholar 

  24. Sontag, E.D. Remarks on feedforward circuits, adaptation, and pulse memory. IET Syst. Biol. 4, 39–51 (2010).

    Article  CAS  Google Scholar 

  25. Bleris, L. et al. Synthetic incoherent feedforward circuits show adaptation to the amount of their genetic template. Mol. Syst. Biol. 7, 519 (2011).

    Article  Google Scholar 

  26. Weber, W., Kramer, B.P. & Fussenegger, M. A genetic time-delay circuitry in mammalian cells. Biotechnol. Bioeng. 98, 894–902 (2007).

    Article  CAS  Google Scholar 

  27. Dunlop, M.J., Cox, R.S., Levine, J.H., Murray, R.M. & Elowitz, M.B. Regulatory activity revealed by dynamic correlations in gene expression noise. Nat. Genet. 40, 1493–1498 (2008).

    Article  CAS  Google Scholar 

  28. Hausser, J. et al. Timescales and bottlenecks in miRNA-dependent gene regulation. Mol. Syst. Biol. 9, 711 (2013).

    Article  CAS  Google Scholar 

  29. Hooshangi, S., Thiberge, S. & Weiss, R. Ultrasensitivity and noise propagation in a synthetic transcriptional cascade. Proc. Natl. Acad. Sci. USA 102, 3581–3586 (2005).

    Article  CAS  Google Scholar 

  30. Schnütgen, F. et al. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat. Biotechnol. 21, 562–565 (2003).

    Article  Google Scholar 

  31. Weber, W. et al. Macrolide-based transgene control in mammalian cells and mice. Nat. Biotechnol. 20, 901–907 (2002).

    Article  CAS  Google Scholar 

  32. Matsuda, T. & Cepko, C.L. Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl. Acad. Sci. USA 104, 1027–1032 (2007).

    Article  CAS  Google Scholar 

  33. Singh, J. & Padgett, R.A. Rates of in situ transcription and splicing in large human genes. Nat. Struct. Mol. Biol. 16, 1128–1133 (2009).

    Article  CAS  Google Scholar 

  34. Leisner, M., Bleris, L., Lohmueller, J., Xie, Z. & Benenson, Y. Rationally designed logic integration of regulatory signals in mammalian cells. Nat. Nanotechnol. 5, 666–670 (2010).

    Article  CAS  Google Scholar 

  35. Kanegae, Y. et al. High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector. Nucleic Acids Res. 39, e7 (2011).

    Article  Google Scholar 

  36. Andrade-Rozental, A.F. et al. Gap junctions: the “kiss of death” and the “kiss of life”. Brain Res. Brain Res. Rev. 32, 308–315 (2000).

    Article  CAS  Google Scholar 

  37. Mesnil, M., Piccoli, C., Tiraby, G., Willecke, K. & Yamasaki, H. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins. Proc. Natl. Acad. Sci. USA 93, 1831–1835 (1996).

    Article  CAS  Google Scholar 

  38. Mohr, L. et al. Gene therapy of hepatocellular carcinoma in vitro and in vivo in nude mice by adenoviral transfer of the Escherichia coli purine nucleoside phosphorylase gene. Hepatology 31, 606–614 (2000).

    Article  CAS  Google Scholar 

  39. Mercer, K.E., Ahn, C.E., Coke, A., Compadre, C.M. & Drake, R.R. Mutation of herpesvirus thymidine kinase to generate ganciclovir-specific kinases for use in cancer gene therapies. Protein Eng. 15, 903–911 (2002).

    Article  CAS  Google Scholar 

  40. Backman, K., Oconnor, M.J., Maruya, A. & Erfle, M. Use of synchronous site-specific recombination in vivo to regulate gene-expression. Bio/Technology 2, 1045–1049 (1984).

    Article  Google Scholar 

  41. Dale, E.C. & Ow, D.W. Gene-transfer with subsequent removal of the selection gene from the host genome. Proc. Natl. Acad. Sci. USA 88, 10558–10562 (1991).

    Article  CAS  Google Scholar 

  42. Benenson, Y. Biomolecular computing systems: principles, progress and potential. Nat. Rev. Genet. 13, 455–468 (2012).

    Article  CAS  Google Scholar 

  43. Ham, T.S., Lee, S.K., Keasling, J.D. & Arkin, A.P. Design and construction of a double inversion recombination switch for heritable sequential genetic memory. PLoS ONE 3, e2815 (2008).

    Article  Google Scholar 

  44. Friedland, A.E. et al. Synthetic gene networks that count. Science 324, 1199–1202 (2009).

    Article  CAS  Google Scholar 

  45. Siuti, P., Yazbek, J. & Lu, T.K. Synthetic circuits integrating logic and memory in living cells. Nat. Biotechnol. 31, 448–452 (2013).

    Article  CAS  Google Scholar 

  46. Bonnet, J., Yin, P., Ortiz, M.E., Subsoontorn, P. & Endy, D. Amplifying genetic logic gates. Science 340, 599–603 (2013).

    Article  CAS  Google Scholar 

  47. Prochazka, L., Angelici, B., Haefliger, B. & Benenson, Y. Highly modular bow-tie gene circuits with programmable dynamic behaviour. Nat. Commun. 5, 4729 (2014).

    Article  CAS  Google Scholar 

  48. Weber, W., Kramer, B.P., Fux, C., Keller, B. & Fussenegger, M. Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells. J. Gene Med. 4, 676–686 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The work was funded the US National Institutes of Health and National Cancer Institute grant 5R01CA155320 and a European Research Council starting grant CellControl. We wish to thank the M. Fussenegger lab in our department for the ET-ETR inducible system, K. Hoelle and L. Prochazka for plasmids and Benenson lab members for discussions. We thank M. Dessing and V. Jaeggin for assistance with flow cytometry and T. Horn for help with imaging. We thank R. Kellogg from S. Tay's laboratory in our department for help with viability assays.

Author information

Authors and Affiliations

  1. Department of Biosystems Science and Engineering, Swiss Federal Institute of Technology Zurich (ETHZ), Basel, Switzerland

    Nicolas Lapique & Yaakov Benenson

Authors
  1. Nicolas Lapique
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yaakov Benenson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.L. conceived of the project, designed experiments, performed all the experiments, analyzed data and wrote the paper. Y.B. conceived of and supervised the project, designed experiments, analyzed data and wrote the paper.

Corresponding author

Correspondence to Yaakov Benenson.

Ethics declarations

Competing interests

Declaration: The results of this manuscript have been submitted as a priority filing to the European Patent office under filing number EP 14001960.5 with the title `Near-perfect digital switching in a synthetic biosensor circuit achieved through temporal control of circuit's genetic makeup.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–19 and Supplementary Tables 1–16. (PDF 26817 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lapique, N., Benenson, Y. Digital switching in a biosensor circuit via programmable timing of gene availability. Nat Chem Biol 10, 1020–1027 (2014). https://doi.org/10.1038/nchembio.1680

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1680

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer