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. Access through your institution Buy or subscribe This is a preview of subscription

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CLONING SYSTEM TO ASSEMBLE MULTIPLE FRAGMENTS Article Open access 25 October 2024 A FRAMEWORK TO EFFICIENTLY DESCRIBE AND SHARE REPRODUCIBLE DNA MATERIALS AND CONSTRUCTION PROTOCOLS Article

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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 AUTHORS AND AFFILIATIONS * Graduate Program in Bioinformatics, Boston

University, Boston, Massachusetts, USA Evan Appleton & Douglas Densmore * Center of Synthetic Biology, Boston University, Boston, Massachusetts, USA Evan Appleton, Traci Haddock & 

Douglas Densmore * Bioinformatics and Systems Biology Graduate Program, University of California, San Diego, La Jolla, California, USA Jenhan Tao * Department of Electrical and Computer

Engineering, Boston University, Boston, Massachusetts, USA Douglas Densmore Authors * Evan Appleton View author publications You can also search for this author inPubMed Google Scholar *

Jenhan Tao View author publications You can also search for this author inPubMed Google Scholar * Traci Haddock View author publications You can also search for this author inPubMed Google

Scholar * Douglas Densmore View author publications You can also search for this author inPubMed Google Scholar 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 SOURCE DATA TO FIG. 1 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE 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 * Received: 27 November 2013 * Accepted: 07 April 2014 * Published:

28 April 2014 * Issue Date: June 2014 * DOI: https://doi.org/10.1038/nmeth.2939 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable

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