New England Biolabs funded the work and paid the salaries of all authors. Lohman are employees of New England Biolabs, a manufacturer and vendor of molecular biology reagents including DNA ligases and Type IIS restriction enzymes. The specific roles of these authors are articulated in the ‘author contributions’ section.Ĭompeting interests: John M. The funder provided support in the form of salaries for: J.M.P., V.P., R.B.K., K.B., E.J.C., and G.J.S.L., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was funded internally by New England Biolabs, Ipswich, MA, of which all authors are employees. Received: ApAccepted: AugPublished: September 2, 2020Ĭopyright: © 2020 Pryor et al. PLoS ONE 15(9):Įditor: Ruslan Kalendar, University of Helsinki, FINLAND MoClo) as well as the formation of robust new high-fidelity standards.Ĭitation: Pryor JM, Potapov V, Kucera RB, Bilotti K, Cantor EJ, Lohman GJS (2020) Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. Full implementation of the tools developed here enables direct expansion of existing assembly standards for modular cloning systems (e.g. 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. These webtools can be used to create customized assemblies from a target DNA sequence or a desired number of fragments. Next, we incorporated these findings into a suite of webtools that design assembly reactions using the experimental data. 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. 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. However, the utility of this methodology has been limited by a lack of resources to guide experimental design. 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. DNA assembly is an integral part of modern synthetic biology, as intricate genetic engineering projects require robust molecular cloning workflows.
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