Skip to main content

Assembly of Complex Pathways Using Type IIs Restriction Enzymes

  • Protocol
  • First Online:
Microbial Metabolic Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1927))

Abstract

Efficient DNA assembly methods are essential tools for synthetic biology and metabolic engineering. Among several recently developed methods that allow assembly of multiple DNA fragments in a single step, DNA assembly using type IIS enzymes provides many advantages for complex pathway engineering. In particular, it provides the ability for the user to quickly assemble multigene constructs using a series of simple one-pot assembly steps starting from libraries of cloned and sequenced parts. We describe here a protocol for assembly of multigene constructs using the modular cloning system (MoClo). Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. Basic parts that are not yet available need to be made. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3:e3647

    Article  Google Scholar 

  2. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One 6:e16765

    Article  CAS  Google Scholar 

  3. Binder A, Lambert J, Morbitzer R, Popp C, Ott T, Lahaye T, Parniske M (2014) A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants. PLoS One 9:e88218

    Article  Google Scholar 

  4. Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P, Fernandez-del-Carmen A, Granell A, Orzaez D (2011) GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One 6:e21622

    Article  CAS  Google Scholar 

  5. Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D (2013) GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol 162:1618–1631

    Article  CAS  Google Scholar 

  6. Lampropoulos A, Sutikovic Z, Wenzl C, Maegele I, Lohmann JU, Forner J (2013) GreenGate—a novel, versatile, and efficient cloning system for plant transgenesis. PLoS One 8:e83043

    Article  Google Scholar 

  7. Emami S, Yee MC, Dinneny JR (2013) A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology. Front Plant Sci 4:339

    Article  Google Scholar 

  8. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S (2012) Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioengineered bugs 3:38–43

    Article  Google Scholar 

  9. Terfruchte M, Joehnk B, Fajardo-Somera R, Braus GH, Riquelme M, Schipper K, Feldbrugge M (2014) Establishing a versatile Golden Gate cloning system for genetic engineering in fungi. Fungal Genet Biol 62:1–10

    Article  Google Scholar 

  10. Kakui Y, Sunaga T, Arai K, Dodgson J, Ji L, Csikasz-Nagy A, Carazo-Salas R, Sato M (2015) Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe. Open Biol 5:150054

    Article  Google Scholar 

  11. Lee ME, DeLoache WC, Cervantes B, Dueber JE (2015) A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth Biol 4:975–986

    Article  CAS  Google Scholar 

  12. Mitchell LA, Chuang J, Agmon N, Khunsriraksakul C, Phillips NA, Cai Y, Truong DM, Veerakumar A, Wang Y, Mayorga M et al (2015) Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic Acids Res 43:6620–6630

    Article  CAS  Google Scholar 

  13. Agmon N, Mitchell LA, Cai Y, Ikushima S, Chuang J, Zheng A, Choi WJ, Martin JA, Caravelli K, Stracquadanio G et al (2015) Yeast golden gate (yGG) for the efficient assembly of S. cerevisiae transcription units. ACS Synth Biol 4:853–859

    Article  CAS  Google Scholar 

  14. Iverson SV, Haddock TL, Beal J, Densmore DM (2016) CIDAR MoClo: improved MoClo assembly standard and new E. coli part library enable rapid combinatorial design for synthetic and traditional biology. ACS Synth Biol 5:99–103

    Article  CAS  Google Scholar 

  15. Moore SJ, Lai HE, Kelwick RJ, Chee SM, Bell DJ, Polizzi KM, Freemont PS (2016) EcoFlex: a multifunctional MoClo kit for E. coli synthetic biology. ACS Synth Biol 5:1059–1069

    Article  CAS  Google Scholar 

  16. Marillonnet S, Werner S (2015) Assembly of multigene constructs using golden gate cloning. Methods Mol Biol 1321:269–284

    Article  Google Scholar 

  17. Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JD, Patron NJ, Marillonnet S (2014) A golden gate modular cloning toolbox for plants. ACS Synth Biol 3:839–843

    Article  CAS  Google Scholar 

  18. Engler C, Marillonnet S (2014) In: Valla S, Lale R (eds) DNA cloning and assembly methods, vol 1116. Humana Press, Clifton, pp 119–131

    Chapter  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Sylvestre Marillonnet .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Marillonnet, S., Werner, S. (2019). Assembly of Complex Pathways Using Type IIs Restriction Enzymes. In: Santos, C., Ajikumar, P. (eds) Microbial Metabolic Engineering. Methods in Molecular Biology, vol 1927. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9142-6_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9142-6_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9141-9

  • Online ISBN: 978-1-4939-9142-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics