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.

Interactive assembly algorithms for molecular cloning

Abstract

Molecular biologists routinely clone genetic constructs from DNA segments and formulate plans to assemble them. However, manual assembly planning is complex, error prone and not scalable. We address this problem with an algorithm-driven DNA assembly planning software tool suite called Raven (http://www.ravencad.org/) that produces optimized assembly plans and allows users to apply experimental outcomes to redesign assembly plans interactively. We used Raven to calculate assembly plans for thousands of variants of five types of genetic constructs, as well as hundreds of constructs of variable size and complexity from the literature. Finally, we experimentally validated a subset of these assembly plans by reconstructing four recombinase-based 'genetic counter' constructs and two 'repressilator' constructs. We demonstrate that Raven's solutions are significantly better than unoptimized solutions at small and large scales and that Raven's assembly instructions are experimentally valid.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Example assembly of the repressilator21 with Gibson assembly.
Figure 2: In silico assembly with Raven.
Figure 3: Interactive assembly.

References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    Article  Google Scholar 

  5. 5

    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 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    Endy, D. Foundations for engineering biology. Nature 438, 449–453 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Arkin, A. Setting the standard in synthetic biology. Nat. Biotechnol. 26, 771–774 (2008).

    CAS  Article  Google Scholar 

  9. 9

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

    CAS  Article  Google Scholar 

  10. 10

    Blakes, J. et al. A heuristic for maximizing DNA reuse in synthetic DNA library assembly. ACS Synth. Biol. 10.1021/sb400161v (20 February 2014).

  11. 11

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

    Article  Google Scholar 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

    Lou, C., Stanton, B., Chen, Y.-J., Munsky, B. & Voigt, C.A. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 30, 1137–1142 (2012).

    CAS  Article  Google Scholar 

  14. 14

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

    CAS  Article  Google Scholar 

  15. 15

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

    Article  Google Scholar 

  16. 16

    Tamsir, A., Tabor, J.J. & Voigt, C.A. Robust multicellular computing using genetically encoded NOR gates and chemical 'wires'. Nature 469, 212–215 (2011).

    CAS  Google Scholar 

  17. 17

    Moon, T.S., Lou, C., Tamsir, A., Stanton, B.C. & Voigt, C.A. Genetic programs constructed from layered logic gates in single cells. Nature 491, 249–253 (2012).

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 20

    Bonnet, J., Subsoontorn, P. & Endy, D. Rewritable digital storage in live cells via engineered control or recombination directionality. Proc. Natl. Acad. Sci. USA 109, 8884–8889 (2012).

    CAS  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

    Bhatia, S. & Densmore, D. Pigeon: a design visualizer for synthetic biology. ACS Synth. Biol. 2, 348–350 (2013).

    CAS  Article  Google Scholar 

  23. 23

    Peccoud, J. et al. Essential information for synthetic DNA sequences. Nat. Biotechnol. 29, 22 (2011).

    CAS  Article  Google Scholar 

  24. 24

    Bilitchenko, L. et al. Eugene: a domain specific language for specifying and constraining synthetic biological parts, devices, and systems. PLoS ONE 6, e18882 (2011).

    CAS  Article  Google Scholar 

  25. 25

    Wright, D.A. et al. Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat. Protoc. 1, 1637–1652 (2006).

    Article  Google Scholar 

Download references

Acknowledgements

The authors would like to thank S. Bhatia, N. Hillson, E. Oberortner and V. Vasilev for conversations regarding the algorithm development. We also thank M. Smanski (Massachusetts Institute of Technology), S. Iverson (Boston University) and the Boston University iGEM team for providing samples and for conversations regarding MoClo cloning experiments. We would like to thank the authors of work from which this work was extended and all alpha-testers of the Raven software. Finally, we would like to thank T.K. Lu (Massachusetts Institute of Technology), C. Voigt (Massachusetts Institute of Technology) and D. Endy (Stanford University) for providing samples of the genetic constructs that were used to implement assembly plans. This work has been funded by the Office of Naval Research under grant no. N00014-11-1-0725.

Author information

Affiliations

Authors

Contributions

E.A., J.T. and D.D. developed the algorithms. E.A. and J.T. implemented the algorithms and user interface. E.A. and T.H. designed and performed experiments. T.H. developed standard MoClo protocols and provided materials. E.A., J.T., T.H. and D.D. wrote the paper.

Corresponding author

Correspondence to Douglas Densmore.

Ethics declarations

Competing interests

D.D. is a co-founder of Lattice Automation, Inc, a company that produces biodesign automation software.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Table 1 and Supplementary Note (PDF 12815 kb)

Supplementary Table 2

List of all 4-bp overhang sequences and their reverse complements (*) for modular overhang assignment (XLSX 13 kb)

Supplementary Software

Raven pseudocode and data files (ZIP 241 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Appleton, E., Tao, J., Haddock, T. et al. Interactive assembly algorithms for molecular cloning. Nat Methods 11, 657–662 (2014). https://doi.org/10.1038/nmeth.2939

Download citation

Further reading

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