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

Nature Protocols volume 9, pages 2075–2089 (2014)Cite this article

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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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References

  1. Keasling, J.D. Synthetic biology for synthetic chemistry. ACS Chem. Biol. 3, 64–76 (2008).

    Article  CAS  Google Scholar 

  2. Torella, J.P. et al. Tailored fatty acid synthesis via dynamic control of fatty acid elongation. Proc. Natl. Acad. Sci. 110, 11290–11295 (2013).

    Article  CAS  Google Scholar 

  3. Prather, K.L.J. & Martin, C.H. De novo biosynthetic pathways: rational design of microbial chemical factories. Curr. Opin. Biotechnol. 19, 468–474 (2008).

    Article  Google Scholar 

  4. Savage, D.F., Way, J. & Silver, P.A. Defossiling fuel: How synthetic biology can transform biofuel production. ACS Chem. Biol. 3, 13–16 (2008).

    Article  CAS  Google Scholar 

  5. Khalil, A.S. & Collins, J.J. Synthetic biology: applications come of age. Nat. Rev. Genet. 11, 367–379 (2010).

    Article  CAS  Google Scholar 

  6. Purnick, P.E.M. & Weiss, R. The second wave of synthetic biology: from modules to systems. Nat. Rev. Mol. Cell Biol. 10, 410–422 (2009).

    Article  CAS  Google Scholar 

  7. Bowen, T.A., Zdunek, J.K. & Medford, J.I. Cultivating plant synthetic biology from systems biology. New Phytol. 179, 583–587 (2008).

    Article  Google Scholar 

  8. Lienert, F., Lohmueller, J.J., Garg, A. & Silver, P.A. Synthetic biology in mammalian cells: next-generation research tools and therapeutics. Nat. Rev. Mol. Cell Biol. 15, 95–107 (2014).

    Article  CAS  Google Scholar 

  9. Ruder, W.C., Lu, T. & Collins, J.J. Synthetic biology moving into the clinic. Science 333, 1248–1252 (2011).

    Article  CAS  Google Scholar 

  10. Weber, W. & Fussenegger, M. Emerging biomedical applications of synthetic biology. Nat. Rev. Genet. 13, 21–35 (2012).

    Article  CAS  Google Scholar 

  11. Benenson, Y. Biomolecular computing systems: principles, progress and potential. Nat. Rev. Genet. 13, 455–468 (2012).

    Article  CAS  Google Scholar 

  12. Bugaj, L.J. & Schaffer, D.V. Bringing next-generation therapeutics to the clinic through synthetic biology. Curr. Opin. Chem. Biol. 16, 355–361 (2012).

    Article  CAS  Google Scholar 

  13. Chen, Y.Y., Galloway, K.E. & Smolke, C.D. Synthetic biology: advancing biological frontiers by building synthetic systems. Genome Biol. 13, 240 (2012).

    Article  Google Scholar 

  14. Agapakis, C.M. et al. Insulation of a synthetic hydrogen metabolism circuit in bacteria. J. Biol. Eng. 4, 3 (2010).

    Article  Google Scholar 

  15. Chen, Y.J. et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat. Methods 10, 659–664 (2013).

    Article  CAS  Google Scholar 

  16. Litcofsky, K.D., Afeyan, R.B., Krom, R.J., Khalil, A.S. & Collins, J.J. Iterative plug-and-play methodology for constructing and modifying synthetic gene networks. Nat. Methods 9, 1077–1080 (2012).

    Article  CAS  Google Scholar 

  17. Zelcbuch, L. et al. Spanning high-dimensional expression space using ribosome-binding site combinatorics. Nucleic Acids Res. 41, e98 (2013).

    Article  CAS  Google Scholar 

  18. Esvelt, K.M. & Wang, H.H. Genome-scale engineering for systems and synthetic biology. Mol. Syst. Biol. 9, 641 (2013).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Gibson, D.G. et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220 (2008).

    Article  CAS  Google Scholar 

  21. Gibson, D.G., Smith, H.O., Hutchison, C.A., Venter, J.C. & Merryman, C. Chemical synthesis of the mouse mitochondrial genome. Nat. Methods 7, 901–903 (2010).

    Article  CAS  Google Scholar 

  22. Torella, J.P. et al. Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Res. 42, 681–689 (2014).

    Article  CAS  Google Scholar 

  23. Lienert, F. et al. Two-and three-input TALE-based AND logic computation in embryonic stem cells. Nucleic Acids Res. 41, 9967–9975 (2013).

    Article  CAS  Google Scholar 

  24. Guye, P., Li, Y., Wroblewska, L., Duportet, X. & Weiss, R. Rapid, modular and reliable construction of complex mammalian gene circuits. Nucleic Acids Res. 41, e156 (2013).

    Article  Google Scholar 

  25. Casini, A. et al. One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy. Nucleic Acids Res. 42, e7 (2014).

    Article  CAS  Google Scholar 

  26. Ramon, A. & Smith, H.O. Single-step linker-based combinatorial assembly of promoter and gene cassettes for pathway engineering. Biotechnol. Lett. 33, 549–555 (2011).

    Article  CAS  Google Scholar 

  27. Du, J., Yuan, Y., Si, T., Lian, J. & Zhao, H. Customized optimization of metabolic pathways by combinatorial transcriptional engineering. Nucleic Acids Res. 40, e142 (2012).

    Article  CAS  Google Scholar 

  28. Knight, T.F. Idempotent vector design for standard assembly of biobricks MIT Artificial Intelligence Laboratory http://dspace.mit.edu/handle/1721.1/21168 (2003).

  29. Anderson, J.C. et al. BglBricks: a flexible standard for biological part assembly. J. Biol. Eng. 4, 1 (2010).

    Article  Google Scholar 

  30. Borujeni, A.E., Channarasappa, A.S. & Salis, H.M. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res. 42, 2646–2659 (2014).

    Article  Google Scholar 

  31. 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 

  32. Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4, e5553 (2009).

    Article  Google Scholar 

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

    Article  Google Scholar 

  34. Quan, J. & Tian, J. Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat. Protoc. 6, 242–251 (2011).

    Article  CAS  Google Scholar 

  35. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    Article  CAS  Google Scholar 

  36. Xiong, A.S. et al. PCR-based accurate synthesis of long DNA sequences. Nat. Protoc. 1, 791–797 (2006).

    Article  CAS  Google Scholar 

  37. de Kok, S. et al. Rapid and reliable DNA assembly via ligase cycling reaction (LCR). ACS Synth. Biol. 3, 97–106 (2014).

    Article  CAS  Google Scholar 

  38. Bayer, T.S. et al. Synthesis of methyl halides from biomass using engineered microbes. J. Am. Chem. Soc. 131, 6508–6515 (2009).

    Article  CAS  Google Scholar 

  39. Balibar, C.J. & Walsh, C.T. In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum. Biochemistry 45, 15444–15457 (2006).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Joseph P Torella, Florian Lienert, Christian R Boehm, Jan-Hung Chen, Jeffrey C Way & Pamela A Silver

  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA

    Jeffrey C Way & Pamela A Silver

Authors
  1. Joseph P Torella
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  2. Florian Lienert
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  5. Jeffrey C Way
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Contributions

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.

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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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