The use of Type IIs enzymes for DNA shuffling has been reported before , , , , . The Golden Gate DNA assembly method utilizes a combination of a TypeIIS restriction enzyme and a ligase to assemble the DNA fragments. The net result is the ordered and seamless assembly of DNA fragments in one reaction.
This cloning system is based on the Golden Gate cloning technology, a method that allows highly efficient directional assembly of multiple DNA fragments in a single reaction . Introduction. Golden Gate Assembly workflow for both simple and complex assemblies. To simplify the DNA assembly process, we developed a multi-kingdom (MK) Golden Gate assembly platform for experimental work in species from … We present here a versatile resource for plant biologists comprising a set of cloning vectors and 96 standardized parts to enable Golden Gate construction of multigene constructs for plant transformation. Golden Gate cloning provides a precision module-based cloning technique for facile assembly of multiple genes in one construct. Golden Gate Assembly uses two Type IIS Restriction Enzymes, which cut DNA outside of the actual recognition site for the enzyme. The Golden Gate cloning method provides a perfect fit for dTALE protein engineering because it allows directional and seamless assembly of multiple DNA fragments. 8 TypeIIS enzymes (e.g., BsaI and BsmBI enzymes) are endonucleases that cut outside their recognition sites, creating 4 bp DNA overhangs. Golden Gate Background: Golden Gate cloning is a strategy that allows ‘single-tube’ ordered assembly of a vector (Backbone) and one or more DNA fragments (Parts) into a single, usually circular, construct which is suitable for direct transformation of a bacterial host. Golden Gate Assembly. The application of this cloning protocol to DNA shuffling results in a protocol that we call ‘Golden Gate shuffling’. The accuracy of the assembly is dependent on the length of the overhang sequences. Golden Gate Assembly.
However, assembly of the modules was performed from gel purified pre-digested DNA fragments.
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