Interactive assembly algorithms for molecular cloning

Appleton, Evan; Tao, Jenhan; Haddock, Traci L.; Densmore, Douglas

doi:10.1038/nmeth.2939

Cited by 41 publications

(42 citation statements)

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“…Software tools will increasingly play an important role in DNA assembly, and are already required for the design of parts for modular DNA assembly 54,55,59 and used in the experimental planning and quality control of large and complex projects [67][68][69] . Many of these software tools can also control liquid handling robots and their ability to automate hundreds of complex modular assemblies has been recently demonstrated 62 .…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

“…By formatting parts and protocols according to a standard, future workflows will be set so that parts can be combined efficiently over sequential tiers and exchanged between projects around the world. As these standards are implemented the next logical steps for DNA assembly should recapitulate mature engineering disciplines: enabling software will be developed, automation of the labour will be introduced and ultimately the work of cloning will be commercially outsourced just as it is already done for gene synthesis and DNA sequencing.Software tools will increasingly play an important role in DNA assembly, and are already required for the design of parts for modular DNA assembly 54,55,59 and used in the experimental planning and quality control of large and complex projects [67][68][69] . Many of these software tools can also control liquid handling robots and their ability to automate hundreds of complex modular assemblies has been recently demonstrated 62 .…”

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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: Conclusion and Future Perspectivesmentioning

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

View full text Add to dashboard Cite

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“…Currently, there are automation programs that can significantly reduce error rates and manual planning by optimizing assembly sequence and strategy and that have the potential to be integrated with other systems (e.g., robotics) to automate the whole process. 15,25,33,34 Robotic technology is expensive, however, and the consumable costs (plates, pipet tips, chemicals, cuvettes, etc.) can make assays costly.…”

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confidence: 99%

A Versatile Microfluidic Device for Automating Synthetic Biology

et al. 2015

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New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.

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“…For large assemblies or large sets of assemblies, it is desirable to maximize fragment reuse and minimize cloning steps to minimize time and material expense. Some algorithms have been developed to automate this design process (Densmore et al 2010;Appleton et al 2014a;Blakes et al 2014) and have resulted in two web-based open-source software tools: DNALD (see dnald.org/planner) and Raven (see cidar.bu.edu/ravencad). DNALD inputs sets of target parts made of combinations of "basic part" primitives and determines an optimized hierarchical assembly plan for building all target parts assuming that only two fragments can ever be joined in one cloning step (called "binary assembly").…”

Section: Assembly Planningmentioning

confidence: 99%

Design Automation in Synthetic Biology

Appleton

Madsen

Roehner

et al. 2017

Cold Spring Harb Perspect Biol

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Design automation refers to a category of software tools for designing systems that work together in a workflow for designing, building, testing, and analyzing systems with a target behavior. In synthetic biology, these tools are called bio-design automation (BDA) tools. In this review, we discuss the BDA tools areas-specify, design, build, test, and learn-and introduce the existing software tools designed to solve problems in these areas. We then detail the functionality of some of these tools and show how they can be used together to create the desired behavior of two types of modern synthetic genetic regulatory networks.S ynthetic biology as a discipline has concerned itself with "forward engineering" living systems. This is a purposefully grand goal and is faced with numerous scientific, engineering, political, and ethical challenges. These engineering challenges include the storage of biological information, the design of complex, interacting biological systems using that information, the physical creation of these systems, and the dissemination of foundational principles as abstractions on which the next technical advances can be made. What should be clear to even the most casual observer is that computers, and the computational capabilities they bring with them, are going to be required if the field is going to succeed going forward. Debates remain regarding the framing of the computational approaches (Andrianantoandro et al. 2006;Lux et al. 2012;Densmore and Bhatia 2014), but there are few who debate the important and increasingly prominent role of computation in the field.Synthetic biology and its associated promise have attracted a wide variety of participants. In particular, computer engineers and computer scientists began developing computational tools for engineering these genetic systems using techniques from their disciplines. Electronic design automation (EDA) showed a clear process by which design formalisms could be built, software tools created, and a larger, separate industry developed (see latticeautomation.com; teselagen.com; deskgen.com; benchling.com). This pursuit in the synthetic biology field has recently been coined bio-design automation (BDA) (Densmore 2012). BDA often uses a "divide and conquer" approach for solving small parts of a larger problem one piece at a time and allows for specialists to tackle specific narrow problems in which they have considerable ex-

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Interactive assembly algorithms for molecular cloning

Cited by 41 publications

References 25 publications

Bricks and blueprints: methods and standards for DNA assembly

Bricks and blueprints: methods and standards for DNA assembly

A Versatile Microfluidic Device for Automating Synthetic Biology

Design Automation in Synthetic Biology

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