High‐Complexity One‐Pot Golden Gate Assembly

Abstract Golden Gate Assembly is a flexible method of DNA assembly and cloning that permits the joining of multiple fragments in a single reaction through predefined connections. The method depends on cutting DNA using a Type IIS restriction enzyme, which cuts outside its recognition site and therefore can generate overhangs of any sequence while separating the recognition site from the generated fragment. By choosing compatible fusion sites, Golden Gate permits the joining of multiple DNA fragments in a defined order in a single reaction. Conventionally, this method has been used to join five to eight fragments in a single assembly round, with yield and accuracy dropping off rapidly for more complex assemblies. Recently, we demonstrated the application of comprehensive measurements of ligation fidelity and bias data using data‐optimized assembly design (DAD) to enable a high degree of assembly accuracy for very complex assemblies with the simultaneous joining of as many as 52 fragments in one reaction. Here, we describe methods for applying DAD principles and online tools to evaluate the fidelity of existing fusion site sets and assembly standards, selecting new optimal sets, and adding fusion sites to existing assemblies. We further describe the application of DAD to divide known sequences at optimal points, including designing one‐pot assemblies of small genomes. Using the T7 bacteriophage genome as an example, we present a protocol that includes removal of native Type IIS sites (domestication) simultaneously with parts generation by PCR. Finally, we present recommended cycling protocols for assemblies of medium to high complexity (12‐36 fragments), methods for producing high‐quality parts, examples highlighting the importance of DNA purity and fragment stoichiometric balance for optimal assembly outcomes, and methods for assessing assembly success. © 2023 New England Biolabs, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Assessing the fidelity of an overhang set using the NEBridge Ligase Fidelity Viewer Basic Protocol 2 : Generating a high‐fidelity overhang set using the NEBridge GetSet Tool Alternate Protocol 1 : Expanding an existing overhang set using the NEBridge GetSet Tool Basic Protocol 3 : Dividing a genomic sequence with optimal fusion sites using the NEBridge SplitSet Tool Basic Protocol 4 : One‐pot Golden Gate Assembly of 12 fragments into a destination plasmid Alternate Protocol 2 : One‐pot Golden Gate Assembly of 24+ fragments into a destination plasmid Basic Protocol 5 : One‐pot Golden Gate Assembly of the T7 bacteriophage genome from 12+ parts Support Protocol 1 : Generation of high‐purity amplicons for assembly Support Protocol 2 : Cloning assembly parts into a holding vector Support Protocol 3 : Quantifying DNA concentration using a Qubit 4 fluorometer Support Protocol 4 : Visualizing large assemblies via TapeStation Support Protocol 5 : Validating phage genome assemblies via ONT long‐read sequencing