A Modular Cloning System for Standardized Assembly of Multigene Constructs

Weber, Ernst; Engler, Carola; Gruetzner, Ramona; Werner, Stefan; Marillonnet, Sylvestre

doi:10.1371/journal.pone.0016765

Cited by 1,150 publications

(1,363 citation statements)

References 21 publications

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“…To enable using Golden Gate for hierarchical assembly, a variety of physical standards have been developed that adopt a tiered assembly approach: in the first tier genes are assembled from their elementary parts (promoters, open reading frames, terminators, etc. ; Figure 1a) [29][30][31][32] and then in the second tier, these genes are combined to form multi-gene systems (Figure 1b). The MoClo standard, first developed for plant molecular biology, uses a parallel approach for all tiers but requires the use of a large number of entry and destination vectors 29 .…”

Section: Endonuclease-mediated Assemblymentioning

confidence: 99%

“…; Figure 1a) [29][30][31][32] and then in the second tier, these genes are combined to form multi-gene systems (Figure 1b). The MoClo standard, first developed for plant molecular biology, uses a parallel approach for all tiers but requires the use of a large number of entry and destination vectors 29 . The GoldenBraid standard reduces the number of required vectors by applying a pairwise approach for assembly beyond the gene-level, but at the cost of requiring more steps for larger constructs 30,33 .…”

Section: Endonuclease-mediated Assemblymentioning

confidence: 99%

“…Simultaneous digestion and ligation forces incorrectly assembled plasmids to be recut by BsaI while correct ones are protected. (b) Diagram of the MoClo and GoldenBraid 2.0 standards 29,30 . These provide two different approaches for the second tier of Golden Gate assembly, where the genes built in the first tier reaction are combined to form multi-gene constructs.…”

Section: Box 1: Genome Editingmentioning

confidence: 99%

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Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

251

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: Endonuclease-mediated Assemblymentioning

confidence: 99%

Section: Endonuclease-mediated Assemblymentioning

confidence: 99%

Section: Box 1: Genome Editingmentioning

confidence: 99%

See 1 more Smart Citation

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

251

186

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“…Many advances have been achieved towards the design of novel genetic and 21 metabolic pathways of increasing complexity in the past decade [1][2] [3]. In recent years, research 22 has also been devoted to design considerations where predictability is a challenge, such as situations 23…”

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confidence: 99%

Overloaded and stressed: whole-cell considerations for bacterial synthetic biology

Borkowski

Ceroni

Stan

et al. 2016

Current Opinion in Microbiology

235

215

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“…Illustrating these and the strengths of regulation present is a challenge as the complexity of a circuit grows. Similarly, the construction of large variant libraries that explore a potential 5 genetic design space has become commonplace as DNA synthesis costs have fallen and assembly methods have improved [16][17][18][19] . Visualizing a large number of internal circuit states or design variants manually is both time-consuming and highly error-prone.…”

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confidence: 99%

DNAplotlib: Programmable Visualization of Genetic Designs and Associated Data

et al. 2016

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General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms Keywords: visualization; standardization; SBOLv; bio-design automation; synthetic biology 2 Abstract DNAplotlib (www.dnaplotlib.org) is a computational toolkit for the programmable visualization of highly customizable, standards-compliant genetic designs. Functions are provided to aid with both visualization tasks and to extract and overlay associated experimental data. High-quality output is produced in the form of vector-based PDFs, rasterized images, and animated movies. All aspects of the rendering process can be easily customized or extended by the user to cover new forms of genetic part or regulation. DNAplotlib supports improved communication of genetic design information and offers new avenues for static, interactive and dynamic visualizations that map and explore the links between the structure and function of genetic parts, devices and systems; including metabolic pathways and genetic circuits. DNAplotlib is crossplatform software developed using Python and released under the MIT license. 3Engineering disciplines rely on standardized pictorial representations of parts and their interconnections to create schematics that clearly communicate how they are pieced together and to enable the reliable construction of large complex systems. In bioengineering, DNA sequences are often synthesized to create genetic constructs that probe or perturb the natural function of a cell or implement novel capabilities. Unlike more established engineering disciplines, the way that a genetic design is visually represented can vary significantly between labs and across different areas of the field. This leads to ambiguities that hinder data exchange, understanding, and the effective reuse of this research.The Synthetic Biology Open Language Visual 1 (SBOLv) initiative was started to help alleviate this problem, defining a set of agreed symbols for commonly used genetic elements. In addition, other schemes such as the Systems Biology Graphical Notation 2 (SBGN) have been developed to more broadly standardize the graphical notation used to describe biological processes. The importance of these standardized approaches has also been recognized by publishers, with a major synthetic biology journal (ACS Synthetic Biology) adopting the use of SBOLv symbols when presenting genetic design information 3 .Although these initiatives will help accelerate adoption of these standards, they rely on the availability of supporting tools to enable the production of compliant diagrams. Some tools do exist to generate SBOLv visualizations from genetic design information, either through graphical point-and-click interfaces (e.g., VectorNTI 4 , TinkerCell 5 , GenoCAD 6 , DeviceEditor 7 and SBOL Designer) or text-based inputs (e.g., Pigeon 8 and VisBOL 9 ). These are effective for small numbers of constructs, but lack the ...

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A Modular Cloning System for Standardized Assembly of Multigene Constructs

Cited by 1,150 publications

References 21 publications

Bricks and blueprints: methods and standards for DNA assembly

Bricks and blueprints: methods and standards for DNA assembly

Overloaded and stressed: whole-cell considerations for bacterial synthetic biology

DNAplotlib: Programmable Visualization of Genetic Designs and Associated Data

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