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Unique nucleotide sequence–guided assembly of repetitive DNA parts for synthetic biology applications

Abstract

Recombination-based DNA construction methods, such as Gibson assembly, have made it possible to easily and simultaneously assemble multiple DNA parts, and they hold promise for the development and optimization of metabolic pathways and functional genetic circuits. Over time, however, these pathways and circuits have become more complex, and the increasing need for standardization and insulation of genetic parts has resulted in sequence redundancies—for example, repeated terminator and insulator sequences—that complicate recombination-based assembly. We and others have recently developed DNA assembly methods, which we refer to collectively as unique nucleotide sequence (UNS)–guided assembly, in which individual DNA parts are flanked with UNSs to facilitate the ordered, recombination-based assembly of repetitive sequences. Here we present a detailed protocol for UNS-guided assembly that enables researchers to convert multiple DNA parts into sequenced, correctly assembled constructs, or into high-quality combinatorial libraries in only 2–3 d. If the DNA parts must be generated from scratch, an additional 2–5 d are necessary. This protocol requires no specialized equipment and can easily be implemented by a student with experience in basic cloning techniques.

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Figure 1: Overview of UNS-guided assembly.
Figure 2: Sequence considerations when using standard part vectors.
Figure 3: Restriction site locations in part and destination vectors.
Figure 4: Construction of individual circuits and combinatorial libraries.

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Acknowledgements

The authors thank C.T. Walsh, T.A. Wencewicz, P. Malkus and J. Paulsson for plasmids and reagents, and T.J. Ford, D.C. MacKellar, A.H. Chen and H.M. Salis for helpful discussions relating to this work. This work was supported by the Advanced Research Projects Agency-Energy 'Electrofuels' Collaborative Agreement (grant no. DE-AR0000079 to P.A.S.); a National Science Foundation Graduate Research Fellowship and Herchel Smith Graduate Research Fellowship to J.P.T.; a European Molecular Biology Organization and Human Frontier Science Program Fellowship to F.L.; a German National Academic Foundation Scholarship to C.R.B.; a Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship to J.-H.C.; and the Defense Advanced Research Projects Agency grant (no. 4500000572 to P.A.S.). This material is based on work supported by the National Science Foundation. Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

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J.P.T. conceived the project; J.P.T., C.R.B., F.L., J.-H.C., P.A.S. and J.C.W. contributed key ideas to the development of the project; P.A.S. and J.C.W. supervised the project; and J.P.T., C.R.B., F.L. and J.-H.C. performed the experiments.

Corresponding author

Correspondence to Pamela A Silver.

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Torella, J., Lienert, F., Boehm, C. et al. Unique nucleotide sequence–guided assembly of repetitive DNA parts for synthetic biology applications. Nat Protoc 9, 2075–2089 (2014). https://doi.org/10.1038/nprot.2014.145

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