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Interactive assembly algorithms for molecular cloning

Nature Methods volume 11, pages 657662 (2014) | Download Citation

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.

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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

  1. Graduate Program in Bioinformatics, Boston University, Boston, Massachusetts, USA.

    • Evan Appleton
    •  & Douglas Densmore
  2. Center of Synthetic Biology, Boston University, Boston, Massachusetts, USA.

    • Evan Appleton
    • , Traci Haddock
    •  & Douglas Densmore
  3. Bioinformatics and Systems Biology Graduate Program, University of California, San Diego, La Jolla, California, USA.

    • Jenhan Tao
  4. Department of Electrical and Computer Engineering, Boston University, Boston, Massachusetts, USA.

    • Douglas Densmore

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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.

Competing interests

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

Corresponding author

Correspondence to Douglas Densmore.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–12, Supplementary Table 1 and Supplementary Note

Excel files

  1. 1.

    Supplementary Table 2

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

Zip files

  1. 1.

    Supplementary Software

    Raven pseudocode and data files

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DOI

https://doi.org/10.1038/nmeth.2939

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