Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system

Sasaki, Yukari; Sone, Takefumi; Yoshida, Shouhei; Yahata, Kazuhide; Hotta, Junko; Chesnut, Jonathan D.; Honda, Takeshi; Imamoto, Fumio

doi:10.1016/j.jbiotec.2003.10.001

Cited by 111 publications

(89 citation statements)

References 8 publications

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“…This method is simple, efficient and reliable, and is widely used for the generation of clone libraries and for expression analysis in eukaryotic systems 39,40 . By synthetically generating four additional orthogonal att recombination sequences, Gateway has also recently been expanded to enable the cloning of multiple parts simultaneously 41 . Similar non-commercial systems have also been developed that use alternative phage integrases, including phiBT1 and phiC31 42,43 .…”

Section: Site-specific Recombinationmentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

243

186

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PrefaceDNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade a plethora of powerful new DNA assembly methods including Gibson assembly, Golden Gate and LCR have been developed. In this Innovation article we discuss these methods and standards such as MoClo, GoldenBraid, MODAL and PaperClip, which have been developed to facilitate a streamlined assembly workflow, aid material exchange, and the creation of modular, reusable DNA parts. IntroductionOur capacity to cut and paste DNA from different sources and to assemble it into gene constructs has been one of the key drivers of biological research and biotechnology over the past four decades. However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies. DNA assembly also enables carrying out projects with more complex experimental needs and is especially valuable for building diverse plasmid libraries and creating multicomponent systems, and has even been used to construct synthetic cells 2 .Addressing the limitations of DNA assembly methods has been one of the key goals of synthetic biology, a scientific discipline focused on the construction and testing of new or redesigned versions of genes, gene networks, pathways and cells 3,4 . In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 . This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them. These physical standards facilitate the re-use of parts between experiments, exchange of parts between research groups and importantly provide modularity in construction. ...

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Section: Site-specific Recombinationmentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

243

186

View full text Add to dashboard Cite

show abstract

“…To assemble multiple DNA fragments in the desired order, an additional four att sites (att3, att4, att5 and att6) have been developed and applied to MultiSite Gateway cloning (Karimi et al, 2007a;Sasaki et al, 2004). Utilization of these att sites (att1-6) expanded the availability of cloning technology for more complex gene construction.…”

Section: The Pgwb Series (Pgwbxx and Pgwb2xx) Based On The Pbi Plasmidmentioning

confidence: 99%

Gateway Vectors for Plant Genetic Engineering: Overview of Plant Vectors, Application for Bimolecular Fluorescence Complementation (BiFC) and Multigene Construction

Tanaka¹,

Kimura²,

Hikino³

et al. 2012

Genetic Engineering - Basics, New Applications and Responsibilities

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“…Only a handful of dedicated vector assembly systems have been developed in the past several years for the assembly of multigene transformation vectors (Cheo et al, 2004;Sasaki et al, 2004;Karimi et al, 2005;Chen et al, 2006Chen et al, , 2010Wakasa et al, 2006). Most of these vector assembly systems have a rather limited capacity and hence have been utilized for the delivery of only a small number (i.e.…”

mentioning

confidence: 99%

“…The system was used to construct several multitransgene binary vectors and led to the production of transgenic plants with eight transgenes (Chen et al, 2010). A number of other recombination-based cloning strategies have also been developed for the construction of multigene plant transformation vectors (Cheo et al, 2004;Sasaki et al, 2004;Karimi et al, 2005;Chen et al, 2006Chen et al, , 2010Wakasa et al, 2006). However, here too, the irreversible nature of the recombination-based reactions does not enable the modification of existing binary vectors, and users of such systems may be required to completely rebuild their transformation vectors to give different gene combinations.…”

mentioning

confidence: 99%

Zinc Finger Nuclease and Homing Endonuclease-Mediated Assembly of Multigene Plant Transformation Vectors

Zeevi¹,

Liang²,

Arieli³

et al. 2011

Plant Physiol.

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Binary vectors are an indispensable component of modern Agrobacterium tumefaciens-mediated plant genetic transformation systems. A remarkable variety of binary plasmids have been developed to support the cloning and transfer of foreign genes into plant cells. The majority of these systems, however, are limited to the cloning and transfer of just a single gene of interest. Thus, plant biologists and biotechnologists face a major obstacle when planning the introduction of multigene traits into transgenic plants. Here, we describe the assembly of multitransgene binary vectors by using a combination of engineered zinc finger nucleases (ZFNs) and homing endonucleases. Our system is composed of a modified binary vector that has been engineered to carry an array of unique recognition sites for ZFNs and homing endonucleases and a family of modular satellite vectors. By combining the use of designed ZFNs and commercial restriction enzymes, multiple plant expression cassettes were sequentially cloned into the acceptor binary vector. Using this system, we produced binary vectors that carried up to nine genes. Arabidopsis (Arabidopsis thaliana) protoplasts and plants were transiently and stably transformed, respectively, by several multigene constructs, and the expression of the transformed genes was monitored across several generations. Because ZFNs can potentially be engineered to digest a wide variety of target sequences, our system allows overcoming the problem of the very limited number of commercial homing endonucleases. Thus, users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs, and since our cloning system is based on ZFN and homing endonucleases, it may be possible to reconstruct other types of binary vectors and adapt our vectors for cloning on multigene vector systems in various binary plasmids.

show abstract

Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system

Cited by 111 publications

References 8 publications

Bricks and blueprints: methods and standards for DNA assembly

Bricks and blueprints: methods and standards for DNA assembly

Gateway Vectors for Plant Genetic Engineering: Overview of Plant Vectors, Application for Bimolecular Fluorescence Complementation (BiFC) and Multigene Construction

Zinc Finger Nuclease and Homing Endonuclease-Mediated Assembly of Multigene Plant Transformation Vectors

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