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Applications of Ligase Fidelity Data & Tools

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Image of the Golden Gate BridgeLigase fidelity data & tools: Enabling new applications in synthetic biology

As the field of synthetic biology advances and evolves, so do the diverse applications of Golden Gate Assembly. Through its work on ligase fidelity, New England Biolabs® (NEB®) is enabling synthetic biology research with Data-optimized Assembly Design (DAD), NEBridge® Ligase Fidelity Tools, and NEBridge® Golden Gate Assembly reagents, which together power high-complexity and high-fidelity Golden Gate Assemblies.


Featured NEB scientist publications

Learn more about NEB’s research on ligase fidelity and the use of Data-optimized Assembly Design (DAD) and Ligase Fidelity Tools in our growing list below.

High-Complexity One-Pot Golden Gate Assembly

Sikkema, A.P. et al. (2023)
New England Biolabs

Presents a variety of protocols for applying NEB Data-optimized Assembly Design (DAD) and ligase fidelity data to Golden Gate Assembly design using the NEBridge Ligase Fidelity Tools. Protocols include evaluating the fidelity of existing fusion site sets and assembly standards, selecting new optimal sets, adding fusion sites to existing assemblies, dividing known sequences at optimal points, removing native Type IIS sites simultaneously with parts generation by PCR, small genome assembly design, medium to high complexity assembly (12-36 fragments) cycling, and more.

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Profiling DNA Ligase Substrate Specificity with a Pacific Biosciences Single-Molecule Real-Time Sequencing Assay

Duckworth, A. T., et al. (2023)
University of Wisconsin-Madison, New England Biolabs

Describes methods for investigating DNA ligase sequence bias and mismatch discrimination using Pacific Biosciences Single-Molecule Real-Time (PacBio SMRT) sequencing technology. The protocols describe substrate synthesis, library preparation, and data analysis methods suitable for measuring fidelity and bias of DNA ligases. 

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Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design

Pryor, J. M., et al. (2022)
New England Biolabs

Successfully assembled the 40 kb T7 bacteriophage genome from up to 52 parts and recovered infectious phage particles after cellular transformation. The assembly protocols and Data-optimized Assembly Design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

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

Pryor, J. M., et al. (2020)
New England Biolabs

Developed a high-throughput DNA sequencing assay to examine reaction outcomes of Golden Gate assembly with T4 DNA ligase and the most used Type IIS restriction enzymes. These findings were incorporated into a suite of webtools that design assembly reactions using the experimental data. By using these tools, one-pot assemblies of up to 35 DNA fragments were carried out, expanding the limits of current assembly systems.

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Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly

Potapov, V., et al. (2018)
New England Biolabs

Used comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity and enabled accurate and efficient assembly of 24-fragments in a single reaction.

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A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining

Potapov, V., et al. (2018)
New England Biolabs

Development of the method for profiling ligase fidelity and bias during DNA end-joining. This method was used to profile the ligation of all three-base 5'-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment.

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Publications from other scientists

Below is a selection of the latest external publications utilizing NEB’s ligase fidelity data and tools.


FRAGLER: A Fragment Recycler Application Enabling Rapid and Scalable Modular DNA Assembly

Oling, D., et al. (2022)

Describes a comprehensive and robust DNA assembly framework to support rapid and cost-efficient construct generation at scale. The modular system combines features of computationally optimized junctions (NEB Data-optimized Assembly Design), Gly/Ser/Ala codon-junctions (EMMA, MoPET) and a novel software application named FRAGLER (FRAGment recycLER) to perform a variety of functions.


Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

Volke, D. C., et al. (2022)
Technical University of Denmark, Universidad Nacional de Córdoba

Developed a widely-applicable, and high-efficiency genome engineering toolset for Gram-negative bacteria. Tailored a CRISPR base editor that enables single-nucleotide resolution manipulations. Incorporated Cas6-mediated processing of guide RNAs in a streamlined protocol for plasmid assembly using Golden Gate Assembly and NEB Data-optimized Assembly Design, supporting multiplex base editing.


PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies

Yuan, G., et al. (2022)
Oak Ridge National Laboratory, Patuakhali Science and Technology University

Developed a novel method, PARA, which allows for the one-step Golden Gate Assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Utilized NEB Data-optimized Assembly Design and ligase fidelity data.


GEM-Gate: A Low-Cost, Flexible Approach to BioBrick Assembly

Bower, C., et al. (2023)
Alma College

Presents a method for synthetic biologists to convert BioBrick parts into parts for Golden Gate Assembly utilizing NEB Data-optimized Assembly Design.


A Modular Hepatitis E Virus Replicon System for Studies on the Role of ORF1-Encoded Polyprotein Domains

Cierniak, F., et al. (2022)
Friedrich-Loeffler-Institut, German Centre for Infection Research

Developed a modular chimeric reporter replicon system based on cell culture-adapted and rabbit-derived Zoonotic hepatitis E virus (HEV) strains. Utilized NEB ligase fidelity data for selection of optimal overhangs in high-efficiency Golden Gate Assembly.


Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Stuttmann, J., et al. (2021)
Martin Luther University Halle-Wittenberg, Julius Kühn-Institute (JKI), Leibniz Institute of Plant Biochemistry

Developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). The toolkit was used to explore the limits of multiplexing in two major model species and provides perspective on how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes. Utilized NEB Ligase Fidelity tools for Golden Gate Assembly. 


Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging

Bell, N. A. W. and J. E. Molloy (2022)
The Francis Crick Institute

Developed a simple and fast technique for making a diverse range of designed DNA structure constructs by combining PCR amplicons and synthetic oligonucleotides using Golden Gate Assembly rules. Demonstrated high yield fabrication of torsionally-constrained duplex DNA up to 10 kb in length and a variety of DNA hairpin structures.


Bottom-Up Design: A Modular Golden Gate Assembly Platform of Yeast Plasmids for Simultaneous Secretion and Surface Display of Distinct FAP Fusion Proteins

Szent-Gyorgyi, C., et al. (2022)
Carnegie Mellon University

Presents the bottom-up design and plasmid synthesis to prepare 10 kb functional yeast secretion and display plasmids that uses an optimized version of Golden Gate Assembly in combination with fluorogen-activating protein reporter technology. Helps address challenging cloning strategies of single plasmids that rely on combinatorial co-expression of a multitude of target and bait fusion reporters useful in projects like library screens. Utilizes NEB Data-optimized Assembly Design principles.


DIGGER-Bac: prediction of seed regions for high-fidelity construction of synthetic small RNAs in bacteria

Philipp, N., et al. (2023)
Justus-Liebig University Giessen, Max-Planck-Institute for Terrestrial Microbiology, Albert-Ludwigs-Universität Freiburg, Philipps-University Marburg

Introduces DIGGER-Bac, a toolbox for Design and Identification of seed regions for Golden Gate assembly and Expression of synthetic sRNAs in Bacteria. The toolbox supports the process of seed region/scaffold identification (SEEDling) and guides the primer design for high-fidelity Golden Gate cloning (G-GArden). Utilizes NEB Data-optimized Assembly Design principles and Ligase Fidelity Tools.


Novel High-Throughput DNA Part Characterization Technique for Synthetic Biology

Bak, S. K., et al. (2022)
Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology (Republic of Korea)  

Presents a novel DNA part characterization technique that increases throughput by combinatorial DNA part assembly, solid plate-based quantitative fluorescence assay for phenotyping, and barcode tagging-based long-read sequencing for genotyping. Using the techniques, forty-four DNA parts (21 promoters and 23 RBSs) were successfully characterized in 72 h without any automated equipment.


Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species

Malci, K., et al. (2022)
University of Edinburgh, ETH Zürich, University of Brasília, University of São Paulo, Newcastle University

A review of the molecular methods and SynBio toolkits developed for S. cerevisiae and other emerging nonconventional yeast species. Includes Golden Gate Assembly and use of NEB Ligase Fidelity Tools.


SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards

Damalas, S. G., et al. (2020)
Wageningen & Research University (Netherlands), National Center of Biotechnology – CSIC (Spain)

Introduced the SEVA 3.1 platform consisting of the SEVA 3.1 vectors and the Golden Gate-based ‘SevaBrick Assembly’. This platform enables the convergence of standard processes between the SEVA platform, the BioBricks and the Type IIS-mediated DNA assemblies to reduce complexity and optimize compatibility between parts and methods. Utilized NEB ligase fidelity data for selection of optimal overhangs in high-efficiency Golden Gate Assembly.


AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots

Bryant, J. A., Jr., et al. (2023)
Virginia Polytechnic Institute and State University

Presents AssemblyTron, an open-source Python package to integrate j5 DNA assembly design software outputs with build implementation in Opentrons liquid handling robotics with minimal human intervention. Demonstrated the versatility of AssemblyTron through several scarless, multipart DNA assemblies, beginning from fragment amplification, and including Golden Gate Assembly.


Polysaccharide II Surface Anchoring, the Achilles' Heel of Clostridioides difficile

Malet-Villemagne, J., et al. (2023)
Université Paris-Saclay, INRAE, Université Paris Cité, CNRS and Université de Lyon

Provides new tools to reveal the role of essential genes in C. difficile and finds potential new targets to combat C. difficile infection. Utilized NEB Golden Gate Assembly techniques and tools.

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