DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways

Shao, Zengyi; Zhao, Hua; Zhao, Huimin

doi:10.1093/nar/gkn991

Cited by 599 publications

(582 citation statements)

References 42 publications

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“…For example, DNA Assembler performs the assembly of metabolic pathways in S. cerevisiae by initially building individual genes from elementary parts using OE-PCR. These genes are built with end-homology so that upon their transformation into yeast cells they recombine to assemble the desired metabolic pathway cluster 53 . In contrast, the MODAL standard (Figure 3b) for modular construction is more flexible and works with methods as diverse as S. cerevisiae in vivo assembly, Gibson assembly and CPEC 9 .…”

Section: Long Overlap-based Assemblymentioning

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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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: Long Overlap-based Assemblymentioning

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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“…Transformations were either performed by heat shock in the presence of lithium acetate, carrier DNA and PEG 3350 (ref. 60), or by electroporation in the presence of sorbitol 9 .…”

Section: Rs)-norlaudanosoline (Nor) (S)-reticuline (Ret) (S)-scoulmentioning

confidence: 99%

“…The DNA assembler technique, which takes advantage of in vivo homologous recombination in yeast 9 , was used for the assembly of the sanguinarine pathway. Promoters, genes and terminators were assembled by incorporating a B50-bp homologous region between the segments.…”

Section: Rs)-norlaudanosoline (Nor) (S)-reticuline (Ret) (S)-scoulmentioning

confidence: 99%

“…Although Saccharomyces cerevisiae has traditionally been used for the biosynthesis of simple molecules derived from central metabolism 7 , advances in recombinant DNA technology streamlining the cloning of large DNA sequences make this microbe an attractive platform for functional characterization of enzymes as well as the reconstitution of complex metabolic pathways 8,9 . When combined with genetic information on an ever-increasing number of species, microbial hosts provide new opportunities for the discovery and production of diverse and complex natural products.…”

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confidence: 99%

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Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae

et al. 2014

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“…In fact, modular and combinatorial assemblies of various genetic segments, particularly the assembly of large DNA fragments without scars by restriction, digestion, and ligation methods are extremely difficult. 24,27 As a result, several in vitro methods, such as circular polymerase extension cloning, 28 sequence and ligation-independent cloning, 29 Gibson assembly method, 30 Ligase Cycling Reaction (LCR), 31 and in vivo methods including DNA assembly with homologous recombination in Saccharomyces cerevisiae (DNA assembler-yeast) 27,32 have been developed recently. The Gibson assembly method is the most well-known method, and involves the generation of singlestranded complementary overhangs by T5 exonuclease and covalent joining by fusion DNA polymerase and Taq DNA ligase.…”

Section: Rapid Assembly Tools Enable Rapid Evolution Of Genes and Genmentioning

confidence: 99%

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

Kang¹,

Zhang

Jin

et al. 2015

Bioengineered

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DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways

Cited by 599 publications

References 42 publications

Bricks and blueprints: methods and standards for DNA assembly

Bricks and blueprints: methods and standards for DNA assembly

Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

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