Skip to main content

Software-Assisted Stacking of Gene Modules Using GoldenBraid 2.0 DNA-Assembly Framework

  • Protocol
  • First Online:
Plant Functional Genomics

Abstract

GoldenBraid (GB) is a modular DNA assembly technology for plant multigene engineering based on type IIS restriction enzymes. GB speeds up the assembly of transcriptional units from standard genetic parts and facilitates the stacking of several genes within the same T-DNA in few days. GBcloning is software-assisted with a set of online tools. The GBDomesticator tool assists in the adaptation of DNA parts to the GBstandard. The combination of GB-adapted parts to build new transcriptional units is assisted by the GB TU Assembler tool. Finally, the assembly of multigene modules is simulated by the GB Binary Assembler. All the software tools are available at www.gbcloning.org. Here, we describe in detail the assembly methodology to create a multigene construct with three transcriptional units for polyphenol metabolic engineering in plants.

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 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. doi:10.1038/nmeth.1318

    Article  CAS  PubMed  Google Scholar 

  2. Geu-Flores F, Nour-Eldin HH, Nielsen MT, Halkier BA (2007) USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res 35(7):e55. doi:10.1093/nar/gkm106

    Article  PubMed Central  PubMed  Google Scholar 

  3. Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10(11):1788–1795

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Zhang H, Jiang T (2010) Synthetic circuits, devices and modules. Protein Cell 1(11):974–978. doi:10.1007/s13238-010-0133-8

    Article  CAS  PubMed  Google Scholar 

  5. Muller KM, Arndt KM (2012) Standardization in synthetic biology. Methods Mol Biol 813:23–43. doi:10.1007/978-1-61779-412-4_2

    Article  PubMed  Google Scholar 

  6. 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(7):e21622. doi:10.1371/journal.pone.0021622

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Sarrion-Perdigones A, Palaci J, Granell A, Orzaez D (2014) Design and construction of multigenic constructs for plant biotechnology using the GoldenBraid cloning strategy. Methods Mol Biol 1116:133–151. doi:10.1007/978-1-62703-764-8_10

    Article  PubMed  Google Scholar 

  8. Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4(5):e5553. doi:10.1371/journal.pone.0005553

    Article  PubMed Central  PubMed  Google Scholar 

  9. 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(3):1618–1631, pp.113.217661 [pii]

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Mathews H, Clendennen SK, Caldwell CG, Liu XL, Connors K, Matheis N, Schuster DK, Menasco DJ, Wagoner W, Lightner J, Wagner DR (2003) Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15(8):1689–1703

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. De Jong WS, Eannetta NT, De Jong DM, Bodis M (2004) Candidate gene analysis of anthocyanin pigmentation loci in the Solanaceae. TAG Theoretical and applied genetics. Theoretische und angewandte Genetik 108(3):423–432. doi:10.1007/s00122-003-1455-1

    Article  PubMed  Google Scholar 

  12. Adato A, Mandel T, Mintz-Oron S, Venger I, Levy D, Yativ M, Dominguez E, Wang Z, De Vos RC, Jetter R, Schreiber L, Heredia A, Rogachev I, Aharoni A (2009) Fruit-surface flavonoid accumulation in tomato is controlled by a SlMYB12-regulated transcriptional network. PLoS Genet 5(12):e1000777. doi:10.1371/journal.pgen.1000777

    Article  PubMed Central  PubMed  Google Scholar 

  13. Shimada S, Otsuki H, Sakuta M (2007) Transcriptional control of anthocyanin biosynthetic genes in the Caryophyllales. J Exp Bot 58(5):957–967, erl256 [pii]

    Article  CAS  PubMed  Google Scholar 

  14. Ballester AR, Molthoff J, de Vos R, Hekkert B, Orzaez D, Fernandez-Moreno JP, Tripodi P, Grandillo S, Martin C, Heldens J, Ykema M, Granell A, Bovy A (2010) Biochemical and molecular analysis of pink tomatoes: deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color. Plant Physiol 152(1):71–84. doi:10.1104/pp. 109.147322

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, Schijlen EG, Hall RD, Bovy AG, Luo J, Martin C (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26(11):1301–1308. doi:10.1038/nbt.1506

    Article  CAS  PubMed  Google Scholar 

  16. Bedoya LC, Martinez F, Orzaez D, Daros JA (2012) Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis. Plant Physiol 158(3):1130–1138, pp.111.192922 [pii]

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42(6):819–832

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We want to thank the COST Action FA1006 for the support in the development of the Web tools. This work was supported by the Spanish Ministry of Economy and Competitiveness (grant no. BIO2010–15384). M. Vazquez-Vilar is recipient of a Junta de Ampliación de Estudios fellowship.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Diego Orzaez .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Vazquez-Vilar, M., Sarrion-Perdigones, A., Ziarsolo, P., Blanca, J., Granell, A., Orzaez, D. (2015). Software-Assisted Stacking of Gene Modules Using GoldenBraid 2.0 DNA-Assembly Framework. In: Alonso, J., Stepanova, A. (eds) Plant Functional Genomics. Methods in Molecular Biology, vol 1284. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2444-8_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2444-8_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2443-1

  • Online ISBN: 978-1-4939-2444-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics