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.

  • Review Article
  • Published:

Foundations for the design and implementation of synthetic genetic circuits

Nature Reviews Genetics volume 13, pages 406–420 (2012)Cite this article

Subjects

Key Points

  • Synthetic gene circuits are designed to implement novel biologic function, including cellular logic, dynamics and complex cellular and multicellular behaviours. A decade after emerging as a discipline, synthetic biology is entering the mainstream of biological research in molecular and systems biology, biotechnology and biomedicine.

  • One important current frontier in synthetic biology is the design and implementation of circuits and networks which are larger and more sophisticated than those of the early years of the field. A formalized design process, which has been essential in other engineering disciplines for scaling to larger systems, is being developed.

  • Design in synthetic biology combines top-down decomposition to break down complex problems into smaller subproblems with known solutions and bottom-up assembly, which combines components such as promoters, genes or higher-order modules into systems that solve the high-level problem.

  • At the level of molecular parts such as transcription factors, sensors and actuators, formalized design requires availability of large and compatible classes of components. Zinc finger and transcription-activator-like effector proteins, synthetic microRNAs and engineered cell surface receptors will each advance the field.

  • At the level of modules, much early work in synthetic biology has created and evaluated basic dynamic network motifs such as switches, oscillators and cascades, and an empirically informed choice of optimal circuit topologies for a given purposes is now possible. Cell–cell communication modules based on quorum sensing have been widely used; establishing similarly versatile modules in eukaryotic cells is now a priority. Eukaryotic signal processing based on protein–protein interactions has also been engineered.

  • A small number of large, sophisticated, integrated synthetic biological systems has been published. They have built on previously well-characterized dynamic modules as well as parts. Top-down decomposition and bottom-up assembly have allowed such reuse.

  • As the field moves forward, part standardization and computational design tools are likely to make the design and implementation of regulatory networks more predictable. New classes of sensors for chemical or optic stimuli, and new classes of actuators such as master regulators of mammalian cellular processes will broaden the scope of synthetic biology.

  • Interactions with the cellular and extracellular context, nongenetic phenomena such as the sensing and actuation of mechanical forces, and complex nonlinear interactions will require strategies for orthogonalization and insulation and may benefit from combining rational design with library selections.

Abstract

Synthetic gene circuits are designed to program new biological behaviour, dynamics and logic control. For all but the simplest synthetic phenotypes, this requires a structured approach to map the desired functionality to available molecular and cellular parts and processes. In other engineering disciplines, a formalized design process has greatly enhanced the scope and rate of success of projects. When engineering biological systems, a desired function must be achieved in a context that is incompletely known, is influenced by stochastic fluctuations and is capable of rich nonlinear interactions with the engineered circuitry. Here, we review progress in the provision and engineering of libraries of parts and devices, their composition into large systems and the emergence of a formal design process for synthetic biology.

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: Design and evolution of phenotypes on rugged landscapes.
Figure 2: Overview of the computer-aided design process.

Similar content being viewed by others

References

  1. 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  PubMed  Google Scholar 

  2. Weiss, R. & Basu, S. The device physics of cellular logic gates. in NSC-1: The First Workshop on Non-Silicon Computing 54–61 (2002).

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

    Article  CAS  PubMed  Google Scholar 

  4. Mukherji, S. & van Oudenaarden, A. Synthetic biology: understanding biological design from synthetic circuits. Nature Rev. Genet. 10, 859–871 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Nandagopal, N. & Elowitz, M. B. Synthetic biology: integrated gene circuits. Science 333, 1244–1248 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ruder, W. C., Lu, T. & Collins, J. J. Synthetic biology moving into the clinic. Science 333, 1248–1252 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Khalil, A. S. & Collins, J. J. Synthetic biology: applications come of age. Nature Rev. Genet. 11, 367–379 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Yizhar, O. et al. Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477, 171–178 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Carr, P. A. & Church, G. M. Genome engineering. Nature Biotech. 27, 1151–1162 (2009).

    Article  CAS  Google Scholar 

  10. Ellis, T., Adie, T. & Baldwin, G. S. DNA assembly for synthetic biology: from parts to pathways and beyond. Integr. Biol. 3, 109–118 (2011).

    Article  CAS  Google Scholar 

  11. Ro, D.-K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009). Optimization of a gene network by simultaneous modification of multiple ribosome binding sites across a bacterial genome is discussed in this paper. It also shows the potential of fast and efficient genome engineering.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Purnick, P. E. M. & Weiss, R. The second wave of synthetic biology: from modules to systems. Nature Rev. Mol. Cell Biol. 10, 410–422 (2009).

    Article  CAS  Google Scholar 

  15. Todd, M. H. Computer-aided organic synthesis. Chem. Soc. Rev. 34, 247–266 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. MacDonald, J. T., Barnes, C., Kitney, R. I., Freemont, P. S. & Stan, G.-B. V. Computational design approaches and tools for synthetic biology. Integr. Biol. 3, 97–108 (2011).

    Article  Google Scholar 

  17. Chandran, D., Bergmann, F. T., Sauro, H. M. & Densmore, D. Design and Analysis of Biomolecular Circuits: Engineering Approaches to Systems and Synthetic Biology 203–224 (Springer, 2011).

    Book  Google Scholar 

  18. Beal, J., Lu, T. & Weiss, R. Automatic compilation from high-level biologically-oriented programming language to genetic regulatory networks. PLoS ONE 6, e22490 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Grünberg, R. & Serrano, L. Strategies for protein synthetic biology. Nucleic Acids Res. 38, 2663–2675 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Martin, A. R. C. et al. Protein folds and functions. Structure 6, 875–884 (1998).

    Article  CAS  PubMed  Google Scholar 

  21. 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 

  22. Choo, Y., Sánchez-García, I. & Klug, A. In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature 372, 642–645 (1994).

    Article  CAS  PubMed  Google Scholar 

  23. Klug, A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu. Rev. Biochem. 79, 213–231 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Moscou, M. J. & Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Voytas, D. F. & Joung, J. K. D. N. A. Binding made easy. Science 326, 1491–1492 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Morbitzer, R., Römer, P., Boch, J. & Lahaye, T. Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. Proc. Natl Acad. Sci. USA 107, 1–6 (2010).

    Article  Google Scholar 

  28. Davidson, E. A. & Ellington, A. D. Synthetic RNA circuits. Nature Chem. Biol. 3, 23–28 (2007).

    Article  CAS  Google Scholar 

  29. Isaacs, F. J., Dwyer, D. J. & Collins, J. J. RNA synthetic biology. Nature Biotech. 24, 545–554 (2006).

    Article  CAS  Google Scholar 

  30. Salis, H. M. Mirsky, E. A. & Voigt, C. A. Automated design of synthetic ribosome binding sites to control protein expression. Nature Biotech. 27, 946–950 (2009). This study uses a physical chemical model of the interaction between the Shine–Dalgarno sequence and the 16S ribosomal RNA for predictive forward design of ribosomal binding sites of desired strength.

    Article  CAS  Google Scholar 

  31. 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 

  32. 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  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  34. Xie, Z., Liu, S. J., Bleris, L. & Benenson, Y. Logic integration of mRNA signals by an RNAi-based molecular computer. Nucleic Acids Res. 38, 2692–2701 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 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). This paper shows that the use of multiple miRNA biomarker sensors and synthetic genetic logic for the specific identification of a particular human cancer cell type.

    Article  CAS  PubMed  Google Scholar 

  36. Lucks, J. B., Qi, L., Mutalik, V. K., Wang, D. & Arkin, A. P. Versatile RNA-sensing transcriptional regulators for engineering genetic networks. Proc. Natl Acad. Sci. USA 108, 8617–8622 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Cho, E. J., Lee, J.-W. & Ellington, A. D. Applications of aptamers as sensors. Annu. Rev. Anal. Chem. 2, 241–264 (2009).

    Article  CAS  Google Scholar 

  38. Famulok, M., Hartig, J. S. & Mayer, G. Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. Chem. Rev. 107, 3715–3743 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Win, M. N. & Smolke, C. D. A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. Proc. Natl Acad. Sci. USA 104, 14283–14288 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Win, M. N. & Smolke, C. D. Higher-order cellular information processing with synthetic RNA devices. Science 322, 456–460 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ausländer, S., Ketzer, P. & Hartig, J. S. A ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression. Mol. Biosyst. 6, 807–814 (2010).

    Article  PubMed  CAS  Google Scholar 

  42. Joyce, G. F. Forty years of in vitro evolution. Angew. Chem. Int. Edn Engl. 46, 6420–6436 (2007).

    Article  CAS  Google Scholar 

  43. Culler, S. J., Hoff, K. G. & Smolke, C. D. Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins. Science 330, 1251–1255 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Vuyisich, M. & Beal, P. A. Controlling protein activity with ligand-regulated RNA aptamers. Chem. Biol. 9, 907–913 (2002).

    Article  CAS  PubMed  Google Scholar 

  45. Hunsicker, A. et al. An RNA aptamer that induces transcription. Chem. Biol. 16, 173–180 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Skerker, J. M. et al. Rewiring the specificity of two-component signal transduction systems. Cell 133, 1043–1054 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  48. Tabor, J. J. Levskaya, A. & Voigt, C. A. Multichromatic control of gene expression in Escherichia coli. J. Mol. Biol. 405, 315–324 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Toettcher, J. E., Voigt, C. A., Weiner, O. D. & Lim, W. A. The promise of optogenetics in cell biology: interrogating molecular circuits in space and time. Nature Methods 8, 35–38 (2011).

    Article  CAS  PubMed  Google Scholar 

  50. Fenno, L., Yizhar, O. & Deisseroth, K. The development and application of optogenetics. Annu. Rev. Neurosci. 34, 389–412 (2010).

    Article  CAS  Google Scholar 

  51. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neurosci. 8, 1263–1268 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. 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 

  53. Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Airan, R. D., Thompson, K. R., Fenno, L. E., Bernstein, H. & Deisseroth, K. Temporally precise in vivo control of intracellular signalling. Nature 458, 1025–1029 (2009).

    Article  CAS  PubMed  Google Scholar 

  55. Gautier, A., Deiters, A. & Chin, J. W. Light-activated kinases enable temporal dissection of signaling networks in living cells. J. Am. Chem. Soc. 133, 2124–2127 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Magnus, C. J. et al. Chemical and genetic engineering of selective ion channel-ligand interactions. Science 333, 1292–1296 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Pei, Y., Rogan, S. C., Yan, F. & Roth, B. L. Engineered GPCRs as tools to modulate signal transduction. Physiology 23, 313–321 (2008).

    Article  CAS  PubMed  Google Scholar 

  58. Dong, S., Rogan, S. C. & Roth, B. L. Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs. Nature Protoc. 5, 561–573 (2010).

    Article  CAS  Google Scholar 

  59. Lim, W. A. Designing customized cell signalling circuits. Nature Rev. Mol. Cell Biol. 11, 393–403 (2010). This paper reviews a series of studies conducted in the Lim group on engineering the dynamics of protein–protein interaction networks in eukaryotic signal processing by protein domain recombination. Although challenging, this is an important complement to the more widespread engineering of transcriptional regulation.

    Article  CAS  Google Scholar 

  60. Burrill, D. R. & Silver, P. A. Making cellular memories. Cell 140, 13–18 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Purcell, O., Savery, N. J., Grierson, C. S. & di Bernardo, M. A comparative analysis of synthetic genetic oscillators. J. R. Soc. Interface 7, 1503–1524 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Atkinson, M. R., Savageau, M. A., Myers, J. T. & Ninfa, A. J. Development of genetic circuitry exhibiting toggle switch or oscillatory behavior in Escherichia coli. Cell 113, 597–607 (2003).

    Article  CAS  PubMed  Google Scholar 

  63. Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Danino, T., Mondragón-Palomino, O., Tsimring, L. & Hasty, J. A synchronized quorum of genetic clocks. Nature 463, 326–330 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Tigges, M., Marquez-Lago, T. T., Stelling, J. & Fussenegger, M. A tunable synthetic mammalian oscillator. Nature 457, 309–312 (2009).

    Article  CAS  PubMed  Google Scholar 

  66. Kramer, B. P. et al. An engineered epigenetic transgene switch in mammalian cells. Nature Biotech. 22, 867–870 (2004).

    Article  CAS  Google Scholar 

  67. 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  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Brenner, K., You, L. & Arnold, F. H. Engineering microbial consortia: a new frontier in synthetic biology. Trends Biotechnol. 26, 483–489 (2008).

    Article  CAS  PubMed  Google Scholar 

  70. Pai, A., Tanouchi, Y., Collins, C. H. & You, L. Engineering multicellular systems by cell-cell communication. Curr. Opin. Biotechnol. 20, 461–470 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. 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  PubMed  Google Scholar 

  72. Liu, C. et al. Sequential establishment of stripe patterns in an expanding cell population. Science 334, 238–241 (2011).

    Article  CAS  PubMed  Google Scholar 

  73. You, L., Cox, R. S., Weiss, R. & Arnold, F. H. Programmed population control by cell-cell communication and regulated killing. Nature 428, 868–871 (2004).

    Article  CAS  PubMed  Google Scholar 

  74. Balagaddé, F. K. et al. A synthetic Escherichia coli predator-prey ecosystem. Mol. Systems Biol. 4, 187 (2008).

    Article  Google Scholar 

  75. Weber, W., Daoud-El Baba, M. & Fussenegger, M. Synthetic ecosystems based on airborne inter- and intrakingdom communication. Proc. Natl Acad. Sci. USA 104, 10435–10440 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Tamsir, A. Tabor, J. J. & Voigt, C. A. Robust multicellular computing using genetically encoded NOR gates and chemical “wires”. Nature 469, 212–215 (2011). References 76 and 100 demonstrate the decomposition of complex biological logic and dynamics into elementary functions which are implemented in single cells and composed via cell–cell communication in a population.

    Article  CAS  PubMed  Google Scholar 

  77. Tabor, J. J. et al. A synthetic genetic edge detection program. Cell 137, 1272–1281 (2009). An integrated system is described in this paper that combines light sensing, photographic inversion and cell–cell communication modules to produce a pigment only along the edges between illuminated and non-illuminated areas of a bacterial culture on solid medium.

    Article  PubMed  PubMed Central  Google Scholar 

  78. Collins, C. H., Leadbetter, J. R. & Arnold, F. H. Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR. Nature Biotech. 24, 708–712 (2006).

    Article  CAS  Google Scholar 

  79. Sturme, M. H. J. et al. Cell to cell communication by autoinducing peptides in gram-positive bacteria. Antonie van Leeuwenhoek 81, 233–243 (2002).

    Article  CAS  PubMed  Google Scholar 

  80. Dunny, G. M. & Leonard, B. A. Cell–cell communication in Gram-positive bacteria. Annu. Rev. Microbiol. 51, 527–564 (1997).

    Article  CAS  PubMed  Google Scholar 

  81. Clarke, E. J. & Voigt, C. A. Characterization of combinatorial patterns generated by multiple two-component sensors in E. coli that respond to many stimuli. Biotechnol. Bioeng. 108, 666–675 (2011).

    Article  CAS  PubMed  Google Scholar 

  82. Ninfa, A. J. Use of two-component signal transduction systems in the construction of synthetic genetic networks. Curr. Opin. Microbiol. 13, 240–245 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Shou, W., Ram, S. & Vilar, J. M. G. Synthetic cooperation in engineered yeast populations. Proc. Natl Acad. Sci. USA 104, 1877–1882 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Weber, W., Schuetz, M., Dénervaud, N. & Fussenegger, M. A synthetic metabolite-based mammalian inter-cell signaling system. Mol. Biosyst. 5, 757–763 (2009).

    Article  CAS  PubMed  Google Scholar 

  85. Weber, W. et al. Gas-inducible transgene expression in mammalian cells and mice. Nature Biotech. 22, 1440–1444 (2004).

    Article  CAS  Google Scholar 

  86. Wang, W.-D., Chen, Z.-T., Kang, B.-G. & Li, R. Construction of an artificial intercellular communication network using the nitric oxide signaling elements in mammalian cells. Exp. Cell Res. 314, 699–706 (2008).

    Article  CAS  PubMed  Google Scholar 

  87. Chen, M.-T. & Weiss, R. Artificial cell–cell communication in yeast Saccharomyces cerevisiae using signaling elements from Arabidopsis thaliana. Nature Biotech. 23, 1551–1555 (2005).

    Article  CAS  Google Scholar 

  88. Park, S.-H. Zarrinpar, A. & Lim, W. A. Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms. Science 299, 1061–1064 (2003).

    Article  CAS  PubMed  Google Scholar 

  89. Dueber, J. E. Mirsky, E. A. & Lim, W. A. Engineering synthetic signaling proteins with ultrasensitive input/output control. Nature Biotech. 25, 660–662 (2007).

    Article  CAS  Google Scholar 

  90. Bashor, C. J. Helman, N. C., Yan, S. & Lim, W. A. Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics. Science 319, 1539–1543 (2008).

    Article  CAS  PubMed  Google Scholar 

  91. Peisajovich, S. G., Garbarino, J. E., Wei, P. & Lim, W. A. Rapid diversification of cell signaling phenotypes by modular domain recombination. Science 328, 368–372 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Alon, U. Network motifs: theory and experimental approaches. Nature Rev. Genet. 8, 450–461 (2007).

    Article  CAS  PubMed  Google Scholar 

  93. Guet, C. C., Elowitz, M. B., Hsing, W. & Leibler, S. Combinatorial synthesis of genetic networks. Science 296, 1466–1470 (2002).

    Article  CAS  PubMed  Google Scholar 

  94. Francois, P., Hakim, V. & Siggia, E. D. Deriving structure from evolution: metazoan segmentation. Mol. Syst. Biol. 3, 154 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  95. François, P. & Hakim, V. Design of genetic networks with specified functions by evolution in silico. Proc. Natl Acad. Sci. USA 101, 580–585 (2004).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  96. Ellis, T., Wang, X. & Collins, J. J. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nature Biotech. 27, 465–471 (2009). This study achieved predictive, systems-level design of sophisticated regulatory dynamics by quantitative experimental characterization of a library of ribosomal binding sites and computational system design.

    Article  CAS  Google Scholar 

  97. Randall, A., Guye, P., Gupta, S., Duportet, X. & Weiss, R. Design and connection of robust genetic circuits. Meth. Enzymol. 497, 159–186 (2011).

    Article  CAS  Google Scholar 

  98. Silva-Rocha, R. & de Lorenzo, V. Noise and robustness in prokaryotic regulatory networks. Annu. Rev. Microbiol. 64, 257–275 (2010).

    Article  CAS  PubMed  Google Scholar 

  99. Balázsi, G., van Oudenaarden, A. & Collins, J. J. Cellular decision making and biological noise: from microbes to mammals. Cell 144, 910–925 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  100. Regot, S. et al. Distributed biological computation with multicellular engineered networks. Nature 469, 207–211 (2011). See the blurb for reference 76.

    Article  CAS  PubMed  Google Scholar 

  101. Del Vecchio, D., Ninfa, A. J. & Sontag, E. D. Modular cell biology: retroactivity and insulation. Mol. Systems Biol. 4, 161 (2008). This paper derives a model of retroactivity, whereby downstream modules can alter upstream dynamics — for example, by sequestration effects — and proposes several potential insulation mechanisms to minimize retroactivity.

    Article  CAS  Google Scholar 

  102. Mukherji, S. et al. MicroRNAs can generate thresholds in target gene expression. Nature Genet. 43, 854–859 (2011).

    Article  CAS  PubMed  Google Scholar 

  103. Nandagopal, N. & Elowitz, M. B. Synthetic biology: integrated gene circuits. Science 333, 1244–1248 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Ye, H., Daoud-El Baba, M., Peng, R.-W. & Fussenegger, M. A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science 332, 1565–1568 (2011). This paper provides an integrated synthetic gene circuit using both synthetic and endogenous modules for a proof-of-concept of a potential gene or cell-based synthetic biomedical therapy.

    Article  CAS  PubMed  Google Scholar 

  105. Anderson, J. C. Clarke, E. J., Arkin, A. P. & Voigt, C. A. Environmentally controlled invasion of cancer cells by engineered bacteria. J. Mol. Biol. 355, 619–627 (2006).

    Article  CAS  PubMed  Google Scholar 

  106. Haseltine, E. L. & Arnold, F. H. Synthetic gene circuits: design with directed evolution. Annu. Rev. Biophys. Biomol. Struct. 36, 1–19 (2007).

    Article  CAS  PubMed  Google Scholar 

  107. Röthlisberger, D. et al. Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195 (2008).

    Article  CAS  PubMed  Google Scholar 

  108. Voigt, C. A., Mayo, S. L., Arnold, F. H. & Wang, Z. G. Computational method to reduce the search space for directed protein evolution. Proc. Natl Acad. Sci. USA 98, 3778–3783 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Lutz, S. & Patrick, W. M. Novel methods for directed evolution of enzymes: quality, not quantity. Curr. Opin. Biotechnol. 15, 291–297 (2004).

    Article  CAS  PubMed  Google Scholar 

  110. Katz, R. H. Contemporary Logic Design. (Benjamin Cummings, 1994).

    Google Scholar 

  111. Corey, E. J. The logic of chemical synthesis: multistep synthesis of complex carbogenic molecules (Nobel Lecture). Angew. Chem. Int. Edn Engl. 30, 455–465 (1991).

    Article  Google Scholar 

  112. Corey, E., Long, A. & Rubenstein, S. Computer-assisted analysis in organic synthesis. Science 228, 408–418 (1985).

    Article  CAS  PubMed  Google Scholar 

  113. Hoogenboom, H. R. Selecting and screening recombinant antibody libraries. Nature Biotech. 23, 1105–1116 (2005).

    Article  CAS  Google Scholar 

  114. Hackel, B. J., Kapila, A. & Wittrup, K. D. Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling. J. Mol. Biol. 381, 1238–1252 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Boersma, Y. L. & Plückthun, A. DARPins and other repeat protein scaffolds: advances in engineering and applications. Curr. Opin. Biotechnol. 22, 849–57 (2011).

    Article  CAS  PubMed  Google Scholar 

  116. Leisner, M., Bleris, L., Lohmueller, J., Xie, Z. & Benenson, Y. Rationally designed logic integration of regulatory signals in mammalian cells. Nature Nanotechnol. 5, 1–5 (2010).

    Article  CAS  Google Scholar 

  117. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nature Biotech. 29, 143–148 (2011).

    CAS  Google Scholar 

  119. Conklin, B. R. et al. Engineering GPCR signaling pathways with RASSLs. Persp. 5, 673–678 (2008).

    CAS  Google Scholar 

  120. Gunaydin, L. a. et al. Ultrafast optogenetic control. Nature Neurosci. 13, 387–392 (2010).

    Article  CAS  PubMed  Google Scholar 

  121. Weissman, K. J. & Leadlay, P. F. Combinatorial biosynthesis of reduced polyketides. Nature Rev. Microbiol. 3, 925–936 (2005).

    Article  CAS  Google Scholar 

  122. Cane, D. E. Harnessing the biosynthetic code: combinations, permutations, and mutations. Science 282, 63–68 (1998).

    Article  CAS  PubMed  Google Scholar 

  123. Jiang, L. et al. De novo computational design of retro-aldol enzymes. Science 319, 1387–1391 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Richter, F., Leaver-Fay, A., Khare, S. D., Bjelic, S. & Baker, D. De novo enzyme design using Rosetta3. PLoS ONE 6, e19230 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Kramer, B. P., Fischer, C. & Fussenegger, M. BioLogic gates enable logical transcription control in mammalian cells. Biotechnol. Bioeng. 87, 478–484 (2004).

    Article  CAS  PubMed  Google Scholar 

  126. Tigges, M., Dénervaud, N., Greber, D., Stelling, J. & Fussenegger, M. A synthetic low-frequency mammalian oscillator. Nucleic Acids Res. 38, 2702–2711 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Swinburne, I., Miguez, D. G., Landgraf, D. & Silver, P. Intron length increases oscillatory periods of gene expression in animal cells. Genes Dev. 22, 2342–2346 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Gerhart, J. & Kirschner, M. The theory of facilitated variation. Proc. Natl Acad. Sci. USA 104, 8582–8589 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Yuh, C. H., Bolouri, H. & Davidson, E. H. Genomic cis-regulatory logic: experimental and computational analysis of a sea urchin gene. Science 279, 1896–1902 (1998).

    Article  CAS  PubMed  Google Scholar 

  130. Green, J. & Langer, R. A combinatorial polymer library approach yields insight into nonviral gene delivery. Acc. Chem. Res. 41, 749–759 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Kauffman, S. A. The Origins of Order: Self-Organization and Selection in Evolution (Oxford Univ. Press, 1993).

    Google Scholar 

  132. Kauffman, S. A. & Weinberger, E. D. The NK model of rugged fitness landscapes and its application to maturation of the immune response. J. Theor. Biol. 141, 211–245 (1989).

    Article  CAS  PubMed  Google Scholar 

  133. Funahashi, A. et al. CellDesigner 3.5: a versatile modeling tool for biochemical networks. Proc. IEEE 96, 1254–1265 (2008).

    Article  Google Scholar 

  134. Pedersen, M. & Phillips, A. Towards programming languages for genetic engineering of living cells. J. R. Soc. Interface 6, S437–S450 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Czar, M. J., Cai, Y. & Peccoud, J. Writing DNA with GenoCAD. Nucleic Acids Res. 37, W40–W47 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Mirschel, S., Steinmetz, K., Rempel, M. & Ginkel, M. PROMOT: modular modeling for systems biology. Bioinformatics 25, 687–689 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Hill, A. D., Tomshine, J. R., Weeding, E. M. B., Sotiropoulos, V. & Kaznessis, Y. N. SynBioSS: the synthetic biology modeling suite. Bioinformatics 24, 2551–2553 (2008).

    Article  CAS  PubMed  Google Scholar 

  138. Chandran, D., Bergmann, F. T. & Sauro, H. M. TinkerCell: modular CAD tool for synthetic biology. J. Biol. Eng. 29, 19 (2009).

    Article  CAS  Google Scholar 

  139. Rodrigo, G., Carrera, J. & Jaramillo, A. Genetdes: automatic design of transcriptional networks. Bioinformatics 23, 1857–1858 (2007). (2009).

    Article  CAS  PubMed  Google Scholar 

  140. Dasika, M. S. & Maranas, C. D. OptCircuit: an optimization based method for computational design of genetic circuits. BMC Systems Biol. 2, 24 (2008).

    Article  CAS  Google Scholar 

  141. Batt, G., Yordanov, B., Weiss, R. & Belta, C. Robustness analysis and tuning of synthetic gene networks. Bioinformatics 23, 2415–2422 (2007).

    Article  CAS  PubMed  Google Scholar 

  142. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Hoops, S. et al. COPASI-a COmplex PAthway SImulator. Bioinformatics 22, 3067–3074 (2006).

    Article  CAS  PubMed  Google Scholar 

  144. Merks, R. & Glazier, J. A cell-centered approach to developmental biology. Physica A 352, 113–130 (2005).

    Article  CAS  Google Scholar 

  145. Villalobos, A., Ness, J. E., Gustafsson, C., Minshull, J. & Govindarajan, S. Gene Designer: a synthetic biology tool for constructing artificial DNA segments. BMC Bioinformat. 7, 285 (2006).

    Article  CAS  Google Scholar 

  146. Richardson, S. M., Wheelan, S. J., Yarrington, R. M. & Boeke, J. D. GeneDesign: rapid, automated design of multikilobase synthetic genes. Genome Res. 16, 550–556 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Xia, B. et al. Developer's and user's guide to Clotho v2.0 A software platform for the creation of synthetic biological systems. Meth. Enzymol. 498, 97–135 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

A.L.S. is pleased to thank the Boehringer Ingelheim Fonds for support through a Ph.D. fellowship. Work in the Weiss laboratory is supported by the US Defense Advanced Research Projects Agency, the US National Institutes of Health and the US National Science Foundation.

Author information

Authors and Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Adrian L. Slusarczyk, Allen Lin & Ron Weiss

  2. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Ron Weiss

Authors
  1. Adrian L. Slusarczyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Allen Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ron Weiss
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ron Weiss.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

Related links

FURTHER INFORMATION

Adrian L. Slusarczyk's homepage

Allen Lin's homepage

Ron Weiss's homepage

Synthetic Biology Center at MIT

iGEM

Glossary

Abstraction

The process of hiding the extraneous details of a specific implementation to highlight the salient and general features of a system or design.

Actuation

The action on the internal or external environment that constitutes the output of a synthetic gene circuit.

TIM barrel

A conserved protein fold named after triose phosphate isomerase (TIM) and shared among many enzymes with widely differing substrate specificities and catalytic activities.

Immunoglobulin fold

A very common protein fold that is based on a β-sandwich. Contains hypervariable loops, which can accommodate almost any sequence and bind a wide variety of partners.

Photocaged unnatural amino acids

Unnatural amino acids containing a photosensitive masking group, which following activation by light reveals a biologically active functional group.

Quorum sensing

Sensing of population density by cell–cell communication.

Oscillators

A circuit with a periodically varying output signal.

Bandpass filters

A circuit that lets through signals within a certain frequency range but not outside it.

Topology

In a network, the set of all connections among nodes. Depending on what the network signifies (for example, molecular binding, genetic regulation or metabolic fluxes), the network topology takes different meanings. For synthetic gene circuits, topology usually refers to regulatory relationships.

Two-component signalling systems

A type of response system commonly found in bacteria and typically consisting of a membrane-bound, sensory histidine kinase and a soluble response regulator.

Signal transduction

The triggering of an intracellular event following detection of an extracellular cue by a transmembrane receptor molecule.

NAND gate

A digital logic gate that implements the logical NAND, or 'NOT AND'. Its output is low when all inputs are high and is otherwise high.

NOR gates

A digital logic gate that implements the logical NOR, or 'NOT OR'. Its output is low when at least one input is high and is otherwise high.

AND gates

Digital logic gates that implement the logical AND. Their output is high when all inputs are high and is otherwise low.

Binary addition with carry

Addition of numbers represented in a base-2 numeral system, where care is taken to carry digits to the left as necessary. For example, 01b + 01b = 10b (in decimal numbers, 1 + 1 = 2).

Emergent

A term used to describe a phenomenon whereby a system is more than the sum of its parts. An emergent property or behaviour is irreducible.

Kinetic parameters

In a mass action kinetic model of biological dynamics, the kinetic parameters are the constants in the differential equations governing the dynamics of a system, such as rate constants and Hill coefficients.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Slusarczyk, A., Lin, A. & Weiss, R. Foundations for the design and implementation of synthetic genetic circuits. Nat Rev Genet 13, 406–420 (2012). https://doi.org/10.1038/nrg3227

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrg3227

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