MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

Da, Lin; O’Callaghan, Christopher A.

doi:10.1093/nar/gky596

Cited by 27 publications

(33 citation statements)

References 28 publications

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“…Domestication refers to the elimination of RE sites present in the DNA sequence before its use in the assembly system. To date, the most commonly used REs have been BsaI, BsmBI and BpiI, which have 6‐bp recognition sites that, although not frequent on average, are regularly encountered in DNA sequences (Lin & O'Callaghan, ). Type IIS REs, such as SapI and AarI, with 7‐bp recognition sites, can be used to lower the probability of finding sites requiring domestication, and are used in the Electra™ (ATUM, Newark, CA, USA) and GeneArt™ (ThermoFisher, Waltham, MA, USA) kits, respectively.…”

Section: Introductionmentioning

confidence: 99%

Loop assembly: a simple and open system for recursive fabrication of DNA circuits

et al. 2019

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High-efficiency methods for DNA assembly have enabled the routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customization, elaborate vector sets or serial manipulations for the different stages of assembly.We have developed Loop assembly based on a recursive approach to DNA fabrication. The system makes use of two Type IIS restriction endonucleases and corresponding vector sets for efficient and parallel assembly of large DNA circuits. Standardized level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods.Loop assembly enables efficient and versatile DNA fabrication for plant transformation. We show the construction of plasmids up to 16 genes and 38 kb with high efficiency (> 80%). We have characterized Loop assembly on over 200 different DNA constructs and validated the fidelity of the method by high-throughput Illumina plasmid sequencing.Our method provides a simple generalized solution for DNA construction with standardized parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

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Section: Introductionmentioning

confidence: 99%

Loop assembly: a simple and open system for recursive fabrication of DNA circuits

et al. 2019

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“…Standardised methods for DNA assembly have gained popularity in recent years, due to their accessibility, reliability and amenability to automation (1)(2)(3). Among these, Type IIS restriction endonuclease-based assembly systems have been adopted in different fields of biology due to their high efficiency and versatility (4)(5)(6)(7)(8)(9)(10)(11)(12).…”

Section: Introductionmentioning

confidence: 99%

Universal Loop assembly (uLoop): open, efficient, and species-agnostic DNA fabrication

Pollak

Matute

Núñez

et al. 2019

Preprint

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Standardised Type IIS DNA assembly methods are becoming essential for biological engineering and research. Although a 'common syntax' has been proposed to enable higher interoperability between DNA libraries, Golden Gate (GG) -based assembly systems remain specific to target organisms. Furthermore, these GG assembly systems become laborious and unnecessarily complicated beyond the assembly of 4 transcriptional units. Here, we describe "universal Loop" (uLoop) assembly, a simple system based on Loop assembly that enables hierarchical fabrication of large DNA constructs (> 30 kb) for any organism of choice. uLoop comprises two sets of four plasmids that are iteratively used as odd and even levels to compile DNA elements in an exponential manner (4 n-1 ). The elements required for transformation/maintenance in target organisms are also assembled as standardised parts, enabling customisation of host-specific plasmids. Thus, this species-agnostic method decouples efficiency of assembly from the stability of vectors in the target organism. As a proof-of-concept, we show the engineering of multi-gene expression vectors in diatoms, yeast, plants and bacteria. These resources will become available through the OpenMTA for unrestricted sharing and open-access. !

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“…Other Type IIS assembly systems are detailed in Table 2, including the recent developments of 3G Assembly (Halleran et al, 2018), BASIC (Storch et al, 2015(Storch et al, , 2017, Loop Assembly (Pollak et al, 2019), MetClo, (Lin et al, 2018), PODAC (Van Hove et al, 2017, Universal Loop Assembly (Pollak et al, 2020), and VEGAS plus yeast Golden Gate (Chuang et al, 2018;Mitchell et al, 2015). These help to address some of the limitations of MoClo and GoldenBraid; for example, BASIC is an idempotent cloning framework for combinatorial assembly that only requires parts to be domesticated for one restriction enzyme, reducing the risk of altering part function.…”

Section: Type Iis Modular Assembly Methodsmentioning

confidence: 99%

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Young

Haines

Storch

et al. 2021

Metabolic Engineering

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Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.

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MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

Cited by 27 publications

References 28 publications

Loop assembly: a simple and open system for recursive fabrication of DNA circuits

Loop assembly: a simple and open system for recursive fabrication of DNA circuits

Universal Loop assembly (uLoop): open, efficient, and species-agnostic DNA fabrication

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

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