Article

GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology

1 Instituto de Biologia Molecular y Celular de Plantas
Plant physiology (Impact Factor: 6.84). 05/2013; 162(3). DOI: 10.1104/pp.113.217661
Source: PubMed

ABSTRACT

Plant Synthetic Biology aims to apply engineering principles to plant genetic design. One strategic requirement of Plant Synthetic Biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here we describe GoldenBraid2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for Plant Synthetic Biology. GB2.0 relies on the use of TypeIIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally-tested, pre-made genetic modules to build frequently-used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of web resources which include a publicly available database, tutorials and a software package that provides in silico simulations and lab protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement Plant Synthetic Biology projects.

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    • "In addition to the enlarged capacity of cloning vectors, progress has also been made to increase the cloning efficiency using an SSR system, Gateway â cloning technology or the recent utilization of the type IIS restriction enzymes (Golden Gate cloning). Many gene assembly systems have been developed in recent years including the Gateway â technology-based gene assembly technology (Chen et al., 2006), GoldenBraid system (Sarrion-Perdigones et al., 2011, 2013), modular cloning (MoClo) system (Weber et al., 2011; Werner et al., 2012), recombination-assisted multifunctional DNA assembly platform (RMDAP) (Ma et al., 2011), and multiple-round in vivo site-specific assembly (MISSA) system (Chen et al., 2010). Interested readers can examine these references for the details of these technologies. "
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    • "Briefly, the first step was the domestication of CsCCD2 and CaCCD2, which comprises the addition of flanking BsaI and BtgZI sites, and the removal of internal BsaI, BsmBI and BtgZI sites in the CCD2 sequences; PCR amplifications of CsCCD2 and CaCCD2 using GB-adapted primers were performed using pGEMT-CsCCD2 and pGEMT-CaCCD2 as templates. Subsequent cloning of the resulting PCR fragments into the pUPD vector was carried out to yield domesticated GB parts using a BsmBI restriction-ligation reaction as described (Sarrion-Perdigones et al., 2013). For the assembly of the domesticated GB parts into a destination plasmid , we chose pDGB2a1 using the following GB "
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    • "These authors have combined various aspects of recombinase technology, PCR, and use of rare restriction site endonucleases to achieve substantial increases in the number of transcription units that can be assembled in the ultimate binary vector, while also controlling cassette orientation and eliminating or reducing 'scars' (additional bp of DNA sequence unrelated to the introduced gene cassettes). These 'HomeRun' and 'GoldenGate' systems, along with variations and improvements thereof (Engler et al. 2008, 2014; Sarrion-Perdigones et al. 2013; Lampropoulos et al. 2013; Binder et al. 2014; Li et al. 2014) offer enormous potential for rapid, robust, and highly flexible 'plug-and-play' modular systems for plant synthetic biology strategies in the near future. However, they have not yet been applied in practice to Fig. 8 Quantification of changes in seed fatty acid composition due to RNAi-mediated gene silencing. "
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Questions & Answers about this publication

  • Alejandro Sarrion-Perdigones added an answer in Kanamycin:
    Where can I find a gateway RNAi vector with Kanamycin resistance as a selection marker?

    This vector I want to construct an RNAi vector through ligation independent cloning of gateway technology.

    Alejandro Sarrion-Perdigones

    I'd suggest you to use type IIs cloning for this, such as the GoldenBraid2.0 Framework. Visit www.gbcloning.org and check the paper I attach. We have a set of ready-to-use tools for many plant biotechnoogy proposes, including RNAi. 

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      [Show abstract] [Hide abstract]
      ABSTRACT: Plant Synthetic Biology aims to apply engineering principles to plant genetic design. One strategic requirement of Plant Synthetic Biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here we describe GoldenBraid2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for Plant Synthetic Biology. GB2.0 relies on the use of TypeIIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally-tested, pre-made genetic modules to build frequently-used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of web resources which include a publicly available database, tutorials and a software package that provides in silico simulations and lab protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement Plant Synthetic Biology projects.
      Full-text · Article · May 2013 · Plant physiology