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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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.
D.D. is a co-founder of Lattice Automation, Inc, a company that produces biodesign automation software.
Supplementary Figures 1–12, Supplementary Table 1 and Supplementary Note (PDF 12815 kb)
List of all 4-bp overhang sequences and their reverse complements (*) for modular overhang assignment (XLSX 13 kb)
Raven pseudocode and data files (ZIP 241 kb)
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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
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