This video discusses Domestication, or the removal of Type IIS cut sites naturally occurring in vector or insert sequences, as it relates to Golden Gate Assembly.
Golden Gate Assembly is a molecular cloning technique used to join multiple DNA fragments together in a single reaction. Golden Gate methods, including MoClo, GoldenBraid 2.0, Mobius Assembly and EMMA, are used by the synthetic biology community to facilitate sharing of standardized parts in assembly. These methods are efficient and cost-effective means to generate recombinant DNA construct variants. The method utilizes Type IIS restriction enzymes, which allow multiple fragments to be assembled in a single reaction – resulting in seamless assembly. It’s important to make sure the Type IIS recognition sites that you are working with are not present in either your vector or insert sequences, excluding, of course, where needed to create the desired fusion sites. If internal sites are present, the enzyme will cut the DNA in your final assembly, leaving you with linear DNA that doesn’t transform. Removal of internal Type IIS restriction sites in order to prevent these assembly problems, is referred to as “Golden Gate Domestication”. Here are 3 ways you can manage internal sites in your DNA to ensure successful assembly. Method 1: Choose a Type IIS restriction enzyme that does not have any internal sites in your sequences. You can easily check by entering your sequence into our free Golden Gate Assembly tool. Method 2: If internal sites exist, you can eliminate them using site-directed mutagenesis to introduce silent mutations that change the recognition site but not the coded amino acid sequence. This is can be accomplished using a kit such as NEB’s Q5 Site-Directed Mutagenesis Kit. Method 3: Alternatively, you can utilize the internal site as a junction between fragments, and PCR primers can introduce the silent mutation into the sequence simultaneously with part generation to eliminate the restriction site. Golden Gate Assembly, MoClo and other similar assembly methods are powerful and efficient tools and can be successfully used if the internal site domestication issue is appropriately addressed. For more information on Type IIS restriction enzymes, Golden Gate Assembly and other tools, please visit www.neb.com/goldengate.