Related Experiment Video
Updated: Jul 15, 2025
08:31
Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
Published on: February 5, 2021
13.6K
High-Complexity One-Pot Golden Gate Assembly
Andrew P Sikkema1, S Kasra Tabatabaei1, Yan-Jiun Lee1
1Research Department, New England Biolabs, Ipswich, Massachusetts, USA.
Current Protocols
|September 27, 2023
Summary
This study enhances DNA assembly using data-optimized design (DAD) for high accuracy in complex Golden Gate Assembly projects. DAD enables joining many DNA fragments efficiently, improving cloning yields and success rates.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Biotechnology
Background:
- Golden Gate Assembly is a versatile DNA cloning method using Type IIS restriction enzymes for joining multiple DNA fragments in a single reaction.
- Traditional Golden Gate Assembly methods face limitations in accuracy and yield when assembling a large number of fragments (typically 5-8).
- Recent advancements have shown the potential for highly complex assemblies, joining up to 52 fragments with improved accuracy.
Purpose of the Study:
- To describe methods for applying Data-Optimized Assembly Design (DAD) principles and online tools to enhance Golden Gate Assembly.
- To evaluate and optimize fusion site sets, divide genomic sequences, and design one-pot assemblies for complex DNA constructs.
- To provide protocols for high-complexity assemblies, including T7 bacteriophage genome assembly, and methods for assessing success.
Main Methods:
- Application of DAD principles and online tools (NEBridge Ligase Fidelity Viewer, GetSet Tool, SplitSet Tool) for evaluating and selecting fusion sites.
- Design and execution of one-pot Golden Gate Assemblies for medium to high complexity (12-36 fragments), including small genome assembly.
- Protocols for generating high-purity DNA parts, quantifying DNA, visualizing assemblies, and validating results using long-read sequencing.
Main Results:
- Demonstrated high assembly accuracy for complex assemblies by joining up to 52 fragments using DAD.
- Developed and presented methods for optimizing fusion sites, dividing genomic sequences, and performing one-pot assemblies.
- Successfully assembled the T7 bacteriophage genome from multiple parts, showcasing the practical application of DAD principles.
Conclusions:
- Data-Optimized Assembly Design (DAD) significantly improves the accuracy and efficiency of complex Golden Gate Assembly.
- The described methods and online tools facilitate the design and execution of challenging DNA assembly projects.
- This approach enables the construction of complex DNA molecules, including entire genomes, with high fidelity.
Related Concept Videos
01:24Oligosaccharide Assembly
2.9K
Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
Multiple sugar molecules that may or may...
2.9K
02:41Protein Complex Assembly
10.6K
Proteins can form homomeric complexes with another unit of the same protein or heteromeric complexes with different types. Most protein complexes self-assemble spontaneously via ordered pathways, while some proteins need assembly factors that guide their proper assembly. Despite the crowded intracellular environment, proteins usually interact with their correct partners and form functional complexes.
Many viruses self-assemble into a fully functional unit using the infected host cell to...
Many viruses self-assemble into a fully functional unit using the infected host cell to...
10.6K