A User’s Guide to Golden Gate Cloning Methods and Standards

Abstract

The continual demand for specialized molecular cloning techniques that suit a broad range of applications has driven the development of many different cloning strategies. One method that has gained significant traction is Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments. This technique has been modularized and standardized, and includes different subfamilies of methods, the most widely adopted of which are the MoClo and Golden Braid standards. Moreover, specialized toolboxes tailored to specific applications or organisms are also available. Still, the quantity and range of assembly methods can constitute a barrier to adoption for new users, and even experienced scientists might find it difficult to discern which tools are best suited toward their goals. In this review, we provide a beginner-friendly guide to Golden Gate assembly, compare the different available standards, and detail the specific features and quirks of commonly used toolboxes. We also provide an update on the state-of-the-art in Golden Gate technology, discussing recent advances and challenges to inform existing users and promote standard practices.

pGG-PIP: A GreenGate (GG) entry vector collection with Plant Immune system Promoters (PIP)

“…into one ordered expression clone to be used for transgenesis (Fig. 1) (Bird et al ., 2022). Among the developed modular cloning methods, the Golden Gate system has emerged as the most widely utilized version, and in 2013 Lampropoulos et al .…”

Protocol for the transformation and engineering of edible algae Arthrospira platensis to generate heterologous protein-expressing strains

“… Note: To simplify high throughput plasmid generation, the shuttle vectors used by the authors include BsaI restriction sites that are compatible with modular Golden Gate cloning. 4 Maps of backbone plasmids used by the authors are indicated in Figure 1 . These plasmids and their nucleotide sequences are available through Addgene.…”

Benchmarking cassette-based deep mutagenesis by Golden Gate assembly

“…While labs have employed these enzymes to manipulate DNA for the last several decades 5,6 , a major development in their use came in 2008 when Engler et al established a cloning method using the Type IIs enzyme BsaI, which they called Golden Gate assembly 7 . Since then, the method's popularity has grown [8][9][10] , due in part to its ability to connect fragments in a specific order and without a restriction scar 11 .…”