2ab assembly: a methodology for automatable, high-throughput assembly of standard biological parts

Leguía, Mariana; Brophy, Jennifer A N; Densmore, Douglas; Asante, Angel; Anderson, Janet S.

doi:10.1186/1754-1611-7-2

Cited by 10 publications

(8 citation statements)

References 27 publications

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Section: Endonuclease-mediated Assemblymentioning

confidence: 99%

“…These sequences enable a repeatable, idempotent assembly process: the ligation of two BioBrick parts produces a new, larger BioBrick part with the same physical format. As the BioBrick standard became adopted, various improvements (for example BglBricks) were developed that improved its flexibility and efficiency [14][15][16] .Although standards such as BioBricks allow parts to be rationally assembled into desired constructs, it is often quicker to modify existing constructs. New standard plasmid formats have thus been developed to facilitate swapping of parts between constructs.…”

mentioning

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%

mentioning

confidence: 99%

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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“…The RBS Calculator is one of the most commonly used tools in the synthetic biology community: it was used in basic research studies to tune the response of a synthetic AND gate [ 9 ], to generate a set of RBSs of graded strengths to evaluate the transcription/translation processes [ 85 ], and to test DNA assembly platforms [ 33 , 35 ], as well as in applied research to optimize biosynthetic pathways [ 86 , 87 ]. Although it was proved to be useful to guide the choice of proper RBS sequences given a downstream gene, its accuracy is limited and additional tools should be developed to improve the predictability of RBSs [ 57 , 81 ].…”

Section: Research Studies and Tools To Support Bottom-up Designmentioning

confidence: 99%

“…As a result, synthetic biologists so far have mainly focused on the definition of biological parts and on their abstraction and standardization, in order to deal with well-defined components with specific function [ 29 ]. This process has brought to the creation of biological parts repositories including DNA parts that can be shared by the scientific community, like the MIT Registry of Standard Biological Parts [ 30 – 32 ], to standardized and easy-to-automate DNA assembly strategies [ 33 – 35 ], and to standard measurement methodologies to share characterization results of parts, like promoters [ 36 , 37 ]. Researchers have also focused on the realization of engineering-inspired functions to learn the complexity that could be reached in a biological context.…”

Section: Introductionmentioning

confidence: 99%

Advances and Computational Tools towards Predictable Design in Biological Engineering

Pasotti

Zucca

2014

Computational and Mathematical Methods in Medicine

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The design process of complex systems in all the fields of engineering requires a set of quantitatively characterized components and a method to predict the output of systems composed by such elements. This strategy relies on the modularity of the used components or the prediction of their context-dependent behaviour, when parts functioning depends on the specific context. Mathematical models usually support the whole process by guiding the selection of parts and by predicting the output of interconnected systems. Such bottom-up design process cannot be trivially adopted for biological systems engineering, since parts function is hard to predict when components are reused in different contexts. This issue and the intrinsic complexity of living systems limit the capability of synthetic biologists to predict the quantitative behaviour of biological systems. The high potential of synthetic biology strongly depends on the capability of mastering this issue. This review discusses the predictability issues of basic biological parts (promoters, ribosome binding sites, coding sequences, transcriptional terminators, and plasmids) when used to engineer simple and complex gene expression systems in Escherichia coli. A comparison between bottom-up and trial-and-error approaches is performed for all the discussed elements and mathematical models supporting the prediction of parts behaviour are illustrated.

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“…The 2ab assembly method is most relevant to the current study. It utilizes in vivo plasmid methylation and recombination of selectable markers to effect one pot, discontinuous ligations of BglBrick parts using Type II restriction enzymes BglII and BamHI ( Leguia et al, 2013 ). It is efficient, requires little labor and amenable to automation.…”

Section: Introductionmentioning

confidence: 99%

Methylase-assisted subcloning for high throughput BioBrick assembly

Matsumura

2020

PeerJ

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The BioBrick standard makes possible iterated pairwise assembly of cloned parts without any depletion of unique restriction sites. Every part that conforms to the standard is compatible with every other part, thereby fostering a worldwide user community. The assembly methods, however, are labor intensive or inefficient compared to some newer ones so the standard may be falling out of favor. An easier way to assemble BioBricks is described herein. Plasmids encoding BioBrick parts are purified from Escherichia coli cells that express a foreign site-specific DNA methyltransferase, so that each is subsequently protected in vitro from the activity of a particular restriction endonuclease. Each plasmid is double-digested and all resulting restriction fragments are ligated together without gel purification. The ligation products are subsequently double-digested with another pair of restriction endonucleases so only the desired insert-recipient vector construct retains the capacity to transform E. coli. This 4R/2M BioBrick assembly protocol is more efficient and accurate than established workflows including 3A assembly. It is also much easier than gel purification to miniaturize, automate and perform more assembly reactions in parallel. As such, it should streamline DNA assembly for the existing community of BioBrick users, and possibly encourage others to join.

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2ab assembly: a methodology for automatable, high-throughput assembly of standard biological parts

Cited by 10 publications

References 27 publications

Bricks and blueprints: methods and standards for DNA assembly

Bricks and blueprints: methods and standards for DNA assembly

Advances and Computational Tools towards Predictable Design in Biological Engineering

Methylase-assisted subcloning for high throughput BioBrick assembly

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