Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

Marillonnet, Sylvestre; Grützner, Ramona

doi:10.1002/cpmb.115

Cited by 67 publications

(76 citation statements)

References 39 publications

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“…DNA assembly methodologies are routinely used in the field of synthetic biology to generate large, complex recombinant DNA constructs from smaller fragments [ 1 ]. Golden Gate assembly is a DNA assembly methodology that has been particularly useful in these applications as it supports assembly of multiple DNA fragments in a single reaction and is amenable to automation [ 2 – 4 ]. Golden Gate assembly utilizes a Type IIS restriction enzyme to generate DNA fragments with compatible overhang sequences, and a DNA ligase to join the fragments together.…”

Section: Introductionmentioning

confidence: 99%

Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design

et al. 2020

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DNA assembly is an integral part of modern synthetic biology, as intricate genetic engineering projects require robust molecular cloning workflows. Golden Gate assembly is a frequently employed DNA assembly methodology that utilizes a Type IIS restriction enzyme and a DNA ligase to generate recombinant DNA constructs from smaller DNA fragments. However, the utility of this methodology has been limited by a lack of resources to guide experimental design. For example, selection of the DNA sequences at fusion sites between fragments is based on broad assembly guidelines or pre-vetted sets of junctions, rather than being customized for a particular application or cloning project. To facilitate the design of robust assembly reactions, we developed a high-throughput DNA sequencing assay to examine reaction outcomes of Golden Gate assembly with T4 DNA ligase and the most commonly used Type IIS restriction enzymes that generate three-base and four-base overhangs. Next, we incorporated these findings into a suite of webtools that design assembly reactions using the experimental data. These webtools can be used to create customized assemblies from a target DNA sequence or a desired number of fragments. Lastly, we demonstrate how using these tools expands the limits of current assembly systems by carrying out one-pot assemblies of up to 35 DNA fragments. Full implementation of the tools developed here enables direct expansion of existing assembly standards for modular cloning systems (e.g. MoClo) as well as the formation of robust new high-fidelity standards.

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Section: Introductionmentioning

confidence: 99%

Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design

et al. 2020

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“…Constructs were made using the modular cloning system MoClo ( Engler et al, 2014 ; Marillonnet and Grutzner, 2020 ). Coding sequences of the betanin biosynthesis genes BvCYP76AD1 and BvDODA1 were amplified by PCR from red beet cDNA and cloned in the level 0 cloning vector pICH41308 (constructs pAGM7535 and pAGM7523, respectively).…”

Section: Methodsmentioning

confidence: 99%

Engineering Betalain Biosynthesis in Tomato for High Level Betanin Production in Fruits

et al. 2021

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Betalains are pigments found in plants of the Caryophyllales order, and include the red-purple betacyanins and the yellow-orange betaxanthins. The red pigment from red beets, betanin, is made from tyrosine by a biosynthetic pathway that consists of a cytochrome P450, a L-DOPA dioxygenase, and a glucosyltransferase. The entire pathway was recently reconstituted in plants that do not make betalains naturally including potato and tomato plants. The amount of betanin produced in these plants was however not as high as in red beets. It was recently shown that a plastidic arogenate dehydrogenase gene involved in biosynthesis of tyrosine in plants is duplicated in Beta vulgaris and other betalain-producing plants, and that one of the two encoded enzymes, BvADHα, has relaxed feedback inhibition by tyrosine, contributing to the high amount of betanin found in red beets. We have reconstituted the complete betanin biosynthetic pathway in tomato plants with or without a BvADHα gene, and with all genes expressed under control of a fruit-specific promoter. The plants obtained with a construct containing BvADHα produced betanin at a higher level than plants obtained with a construct lacking this gene. These results show that use of BvADHα can be useful for high level production of betalains in heterologous hosts. Unlike red beets that produce both betacyanins and betaxanthins, the transformed tomatoes produced betacyanins only, conferring a bright purple-fuschia color to the tomato juice.

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“…[25,26] Based on this property, unique junction sites, and different antibiotic selection schemes, Golden Gate cloning allows high throughput transfer of desired fragments from donor to recipient vectors. [26] Golden Gate syntaxes of unique cloning junctions have been used extensively for robust, directional assembly in a single reaction of over 30 standard parts, which are then flanked by 4-nt cloning junctions. [27,28] The plant virome is composed of highly divergent viruses with polyphyletic origins.…”

Section: Pasinmentioning

confidence: 99%

Oligonucleotide abundance biases aid design of a type IIS synthetic genomics framework with plant virome capacity

Pasin

2021

Biotechnology Journal

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Synthetic genomics-driven dematerialization of genetic resources facilitates flexible hypothesis testing and rapid product development. Biological sequences have compositional biases, which, I reasoned, could be exploited for engineering of enhanced synthetic genomics systems. In proof-of-concept assays reported herein, the abundance of random oligonucleotides in viral genomic components was analyzed and used for the rational design of a synthetic genomics framework with plant virome capacity (Syn-ViP). Type IIS endonucleases with low abundance in the plant virome, as well as Golden Gate and No See'm principles were combined with DNA chemical synthesis for seamless viral clone assembly by one-step digestion-ligation. The framework described does not require subcloning steps, is insensitive to insert terminal sequences, and was used with linear and circular DNA molecules. Based on a digital template, DNA fragments were chemically synthesized and assembled by one-step cloning to yield a scar-free infectious clone of a plant virus suitable for Agrobacterium-mediated delivery. SynViP allowed rescue of a genuine virus without biological material, and has the potential to greatly accelerate biological characterization and engineering of plant viruses as well as derived biotechnological tools. Finally, computational identification of compositional biases in biological sequences might become a common standard to aid scalable biosystems design and engineering. K E Y W O R D SDNA chemical synthesis, Golden Gate cloning, plant virome, type IIS restriction enzyme, viral infectious clone assembly INTRODUCTIONViruses are relatively simple biological entities, with genomic components whose size is compatible both in terms of technical feasibility and costs with current advances in de novo DNA synthesis. [1,2] Synthetic genomics is becoming commonplace for the study and engineering of animal viruses, especially those with medical interest and human pandemic potential. [3][4][5][6][7] In plant virology, synthetic genomics can help to demonstrate correctness of genomic sequences, rescue of viruses that might not be physically available (e.g., ancient or environmental samples), as well as to accelerate virus reverse genetics, host-virus interaction studies, biological characterization of emerging viruses, or engineering of biotechnological devices. [8][9][10][11] Adoption of this powerful approach for plant virology has nonetheless lagged and is reported only in a handful of studies. [9] The first reports of synthetic genomic approaches in plant virology included the use of oligonucleotides to

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Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

Cited by 67 publications

References 39 publications

Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design

Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design

Engineering Betalain Biosynthesis in Tomato for High Level Betanin Production in Fruits

Oligonucleotide abundance biases aid design of a type IIS synthetic genomics framework with plant virome capacity

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