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.

Bricks and blueprints: methods and standards for DNA assembly

Nature Reviews Molecular Cell Biology volume 16, pages 568–576 (2015)Cite this article

Subjects

Abstract

DNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade, a plethora of powerful new DNA assembly methods — including Gibson Assembly, Golden Gate and ligase cycling reaction (LCR) — have been developed. In this Innovation article, we discuss these methods as well as standards such as the modular cloning (MoClo) system, GoldenBraid, modular overlap-directed assembly with linkers (MODAL) and PaperClip, which have been developed to facilitate a streamlined assembly workflow, to aid the exchange of material between research groups and to create modular reusable DNA parts.

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

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Examples of type IIS restriction endonuclease-based methods and standards.
Figure 2: Site-specific recombination methods and standards.
Figure 3: Examples of long-overlap-based assembly methods and standards.

References

  1. Kosuri, S. & Church, G. M. Large-scale de novo DNA synthesis: technologies and applications. Nat. Methods 11, 499–507 (2014).

    Article  CAS  Google Scholar 

  2. 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 

  3. Keasling, J. D. Synthetic biology for synthetic chemistry. ACS Chem. Biol. 3, 64–76 (2008).

    Article  CAS  Google Scholar 

  4. Cameron, D. E., Bashor, C. J. & Collins, J. J. A brief history of synthetic biology. Nat. Rev. Microbiol. 12, 381–390 (2014).

    Article  CAS  Google Scholar 

  5. Czar, M. J., Anderson, J. C., Bader, J. S. & Peccoud, J. Gene synthesis demystified. Trends Biotechnol. 27, 63–72 (2009).

    Article  CAS  Google Scholar 

  6. Annaluru, N. et al. Total synthesis of a functional designer eukaryotic chromosome. Science 344, 55–58 (2014).

    Article  CAS  Google Scholar 

  7. 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 

  8. Chao, R., Yuan, Y. & Zhao, H. Recent advances in DNA assembly technologies. FEMS Yeast Res. 15, 1–9 (2015).

    Article  Google Scholar 

  9. Casini, A. et al. One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy. Nucleic Acids Res. 42, e7 (2014).

    Article  CAS  Google Scholar 

  10. Cardinale, S. & Arkin, A. P. Contextualizing context for synthetic biology — identifying causes of failure of synthetic biological systems. Biotechnol. J. 7, 856–866 (2012).

    Article  CAS  Google Scholar 

  11. Cohen, S. N., Chang, A. C., Boyer, H. W. & Helling, R. B. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl Acad. Sci. USA 70, 3240–3244 (1973).

    Article  CAS  Google Scholar 

  12. Crook, N. C., Freeman, E. S. & Alper, H. S. Re-engineering multicloning sites for function and convenience. Nucleic Acids Res. 39, e92 (2011).

    Article  CAS  Google Scholar 

  13. Shetty, R. P., Endy, D. & Knight, T. F. Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2, 5 (2008).

    Article  Google Scholar 

  14. Norville, J. E. et al. Introduction of customized inserts for streamlined assembly and optimization of BioBrick synthetic genetic circuits. J. Biol. Eng. 4, 17 (2010).

    Article  CAS  Google Scholar 

  15. Anderson, J. C. et al. BglBricks: a flexible standard for biological part assembly. J. Biol. Eng. 4, 1 (2010).

    Article  Google Scholar 

  16. Leguia, M., Brophy, J. A., Densmore, D., Asante, A. & Anderson, J. C. 2ab assembly: a methodology for automatable, high-throughput assembly of standard biological parts. J. Biol. Eng. 7, 2 (2013).

    Article  CAS  Google Scholar 

  17. Litcofsky, K. D., Afeyan, R. B., Krom, R. J., Khalil, A. S. & Collins, J. J. Iterative plug-and-play methodology for constructing and modifying synthetic gene networks. Nat. Methods 9, 1077–1080 (2012).

    Article  CAS  Google Scholar 

  18. Silva-Rocha, R. et al. The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 41, D666–D675 (2013).

    Article  CAS  Google Scholar 

  19. Martínez-García, E., Aparicio, T., Goñi-Moreno, A., Fraile, S. & de Lorenzo, V. SEVA 2.0: an update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities. Nucleic Acids Res. 43, D1183–D1189 (2014).

    Article  Google Scholar 

  20. Marcaida, M. J., Muñoz, I. G., Blanco, F. J., Prieto, J. & Montoya, G. Homing endonucleases: from basics to therapeutic applications. Cell. Mol. Life Sci. 67, 727–748 (2010).

    Article  CAS  Google Scholar 

  21. Liu, J.-K., Chen, W.-H., Ren, S.-X., Zhao, G.-P. & Wang, J. iBrick: a new standard for iterative assembly of biological parts with homing endonucleases. PLoS ONE 9, e110852 (2014).

    Article  Google Scholar 

  22. Li, M. V. et al. HomeRun vector assembly system: a flexible and standardized cloning system for assembly of multi-modular DNA constructs. PLoS ONE 9, e100948 (2014).

    Article  Google Scholar 

  23. Sleight, S. C. & Sauro, H. M. Visualization of evolutionary stability dynamics and competitive fitness of Escherichia coli engineered with randomized multi-gene circuits. ACS Synth. Biol. 2, 519–528 (2013).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  25. 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  Google Scholar 

  26. Kamens, J. The Addgene repository: an international nonprofit plasmid and data resource. Nucleic Acids Res. 43, D1152–D1157 (2015).

    Article  CAS  Google Scholar 

  27. Mutalik, V. K. et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat. Methods 10, 354–360 (2013).

    Article  CAS  Google Scholar 

  28. Linshiz, G. et al. PR-PR: cross-platform laboratory automation system. ACS Synth. Biol. 3, 515–524 (2014).

    Article  CAS  Google Scholar 

  29. Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A modular cloning system for standardized assembly of multigene constructs. PLoS ONE 6, e16765 (2011).

    Article  CAS  Google Scholar 

  30. Sarrion-Perdigones, A. et al. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162, 1618–1631 (2013).

    Article  CAS  Google Scholar 

  31. Binder, A. et al. A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants. PLoS ONE 9, e88218 (2014).

    Article  Google Scholar 

  32. Lampropoulos, A. et al. GreenGate — a novel, versatile, and efficient cloning system for plant transgenesis. PLoS ONE 8, e83043 (2013).

    Article  Google Scholar 

  33. Sarrion-Perdigones, A. et al. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS ONE 6, e21622 (2011).

    Article  CAS  Google Scholar 

  34. Duportet, X. et al. A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic Acids Res. 42, 13440–13451 (2014).

    Article  CAS  Google Scholar 

  35. Sakuma, T., Nishikawa, A., Kume, S., Chayama, K. & Yamamoto, T. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci. Rep. 4, 5400 (2014).

    Article  CAS  Google Scholar 

  36. Chen, W.-H., Qin, Z.-J., Wang, J. & Zhao, G.-P. The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly. Nucleic Acids Res. 41, e93 (2013).

    Article  CAS  Google Scholar 

  37. Blake, W. J. et al. Pairwise selection assembly for sequence-independent construction of long-length DNA. Nucleic Acids Res. 38, 2594–2602 (2010).

    Article  CAS  Google Scholar 

  38. Hartley, J. L., Temple, G. F. & Brasch, M. A. DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795 (2000).

    Article  CAS  Google Scholar 

  39. Alberti, S., Gitler, A. D. & Lindquist, S. A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24, 913–919 (2007).

    Article  CAS  Google Scholar 

  40. Marsischky, G. & LaBaer, J. Many paths to many clones: a comparative look at high-throughput cloning methods. Genome Res. 14, 2020–2028 (2004).

    Article  CAS  Google Scholar 

  41. Sasaki, Y. et al. Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the multisite Gateway system. J. Biotechnol. 107, 233–243 (2004).

    Article  CAS  Google Scholar 

  42. Zhang, L., Zhao, G. & Ding, X. Tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination. Sci. Rep. 1, 141 (2011).

    Article  Google Scholar 

  43. Colloms, S. D. et al. Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination. Nucleic Acids Res. 42, e23 (2013).

    Article  Google Scholar 

  44. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68 (1989).

    Article  CAS  Google Scholar 

  45. Quan, J. & Tian, J. Circular polymerase extension cloning of complex gene libraries and pathways. PLoS ONE 4, e6441 (2009).

    Article  Google Scholar 

  46. Bitinaite, J. et al. USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res. 35, 1992–2002 (2007).

    Article  CAS  Google Scholar 

  47. Annaluru, N. et al. Assembling DNA fragments by USER fusion. Methods Mol. Biol. 852, 77–95 (2012).

    Article  CAS  Google Scholar 

  48. Li, M. Z. & Elledge, S. J. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat. Methods 4, 251–256 (2007).

    Article  CAS  Google Scholar 

  49. Zhang, Y., Werling, U. & Edelmann, W. SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res. 40, e55 (2012).

    Article  CAS  Google Scholar 

  50. Itaya, M., Fujita, K., Kuroki, A. & Tsuge, K. Bottom-up genome assembly using the Bacillus subtilis genome vector. Nat. Methods 5, 41–43 (2008).

    Article  CAS  Google Scholar 

  51. Raymond, C. K., Pownder, T. A. & Sexson, S. L. General method for plasmid construction using homologous recombination. Biotechniques 26, 134–141 (1999).

    Article  CAS  Google Scholar 

  52. Gibson, D. G. et al. One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc. Natl Acad. Sci. USA 105, 20404–20409 (2008).

    Article  CAS  Google Scholar 

  53. Shao, Z., Zhao, H. & Zhao, H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 37, e16 (2009).

    Article  Google Scholar 

  54. Casini, A. et al. R2oDNA designer: computational design of biologically neutral synthetic DNA sequences. ACS Synth. Biol. 3, 525–528 (2014).

    Article  CAS  Google Scholar 

  55. Torella, J. P. et al. Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Res. 42, 681–689 (2013).

    Article  Google Scholar 

  56. Trubitsyna, M., Michlewski, G., Cai, Y., Elfick, A. & French, C. E. PaperClip: rapid multi-part DNA assembly from existing libraries. Nucleic Acids Res. 42, e154 (2014).

    Article  Google Scholar 

  57. de Kok, S. et al. Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synth. Biol. 3, 97–106 (2014).

    Article  CAS  Google Scholar 

  58. Storch, M. et al. BASIC: a new biopart assembly standard provides accurate, single-tier DNA assembly for synthetic biology. ACS Synth. Biol. http://dx.doi.org/10.1021/sb500356d (2015).

  59. Guye, P., Li, Y., Wroblewska, L., Duportet, X. & Weiss, R. Rapid, modular and reliable construction of complex mammalian gene circuits. Nucleic Acids Res. 41, e156 (2013).

    Article  Google Scholar 

  60. Kahl, L. J. & Endy, D. A survey of enabling technologies in synthetic biology. J. Biol. Eng. 7, 13 (2013).

    Article  Google Scholar 

  61. Gibson, D. G., Smith, H. O., Hutchison, C. A., Venter, J. C. & Merryman, C. Chemical synthesis of the mouse mitochondrial genome. Nat. Methods 7, 901–903 (2010).

    Article  CAS  Google Scholar 

  62. Smanski, M. J. et al. Functional optimization of gene clusters by combinatorial design and assembly. Nat. Biotechnol. 32, 1241–1249 (2014).

    Article  CAS  Google Scholar 

  63. Gibson, D. G. et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220 (2008).

    Article  CAS  Google Scholar 

  64. Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010).

    Article  CAS  Google Scholar 

  65. Jovicevic, D., Blount, B. A. & Ellis, T. Total synthesis of a eukaryotic chromosome: redesigning and SCRaMbLE-ing yeast. Bioessays 36, 855–860 (2014).

    Article  CAS  Google Scholar 

  66. Dymond, J. S. et al. Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477, 471–476 (2011).

    Article  CAS  Google Scholar 

  67. Hillson, N. J., Rosengarten, R. D. & Keasling, J. D. j5 DNA assembly design automation software. ACS Synth. Biol. 1, 14–21 (2012).

    Article  CAS  Google Scholar 

  68. Appleton, E., Tao, J., Haddock, T. & Densmore, D. Interactive assembly algorithms for molecular cloning. Nat. Methods 11, 657–662 (2014).

    Article  CAS  Google Scholar 

  69. Densmore, D. et al. Algorithms for automated DNA assembly. Nucleic Acids Res. 38, 2607–2616 (2010).

    Article  CAS  Google Scholar 

  70. Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    Article  CAS  Google Scholar 

  71. Lajoie, M. J. et al. Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013).

    Article  CAS  Google Scholar 

  72. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  Google Scholar 

  73. Pósfai, G. et al. Emergent properties of reduced-genome Escherichia coli. Science 312, 1044–1046 (2006).

    Article  Google Scholar 

  74. Kim, H. & Kim, J.-S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321–334 (2014).

    Article  CAS  Google Scholar 

  75. Horwitz, A. A. et al. Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR–Cas. Cell Syst. http://dx.doi.org/10.1016/j.cels.2015.02.001 (2015).

  76. Groth, A. C. & Calos, M. P. Phage integrases: biology and applications. J. Mol. Biol. 335, 667–678 (2004).

    Article  CAS  Google Scholar 

  77. Torella, J. P. et al. Unique nucleotide sequence-guided assembly of repetitive DNA parts for synthetic biology applications. Nat. Protoc. 9, 2075–2089 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank M. Jamilly for contributing to initial discussions and apologize to those whose work was not discussed owing to space limitations. Research on DNA assembly in the groups of T.E. and G.S.B. is supported by the UK Engineering and Physical Research Council (EPSRC) grant EP/J02175X/1 and EU FP7 grant KBBE.2011.5-289326. M.S. is supported by Marie Curie Intra-European Fellowship 628019.

Author information

Authors and Affiliations

  1. Department of Life Sciences, Centre for Synthetic Biology and Innovation, Imperial College London, London, SW7 2AZ, UK

    Arturo Casini, Marko Storch & Geoffrey S. Baldwin

  2. Department of Bioengineering, Centre for Synthetic Biology and Innovation, Imperial College London, London, SW7 2AZ, UK

    Tom Ellis

Authors
  1. Arturo Casini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marko Storch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Geoffrey S. Baldwin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tom Ellis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Geoffrey S. Baldwin or Tom Ellis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Casini, A., Storch, M., Baldwin, G. et al. Bricks and blueprints: methods and standards for DNA assembly. Nat Rev Mol Cell Biol 16, 568–576 (2015). https://doi.org/10.1038/nrm4014

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrm4014

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research