Generation of MoClo Standard Parts Using Golden Gate Cloning

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

“…Even though hierarchical assembly is one of the core strengths of Golden Gate, it also adds complexity to the method. Just like any other Golden Gate assembly, a hierarchical assembly relies on properly designed cleavage sites, as reviewed elsewhere. ,, Specifically, it requires destination vectors with two pairs of recognition sites, each recognized by a different endonuclease (Figure ): one to insert the intermediate construct (e.g., a transcriptional unit) into the destination vector, and another one to liberate the assembled construct for further assembly rounds (e.g., combining two transcriptional units together). It also requires at least two restriction endonucleases (for example BsaI and BsmBI) and two selection markers (for example kanamycin and spectinomycin) since these must be alternated between different assembly steps.…”

“…The most important concept in Golden Gate assembly is the "proper design" of endonuclease restriction sites (Figure 3), which is thoroughly reviewed elsewhere. 20 These restriction sites make it possible to extract DNA parts from part vectors and insert the assembled construct into destination vectors. Briefly, the restriction site of a Type IIS endonuclease comprises two parts: the recognition sequence, where the enzyme binds DNA; and the cleavage site, where the enzyme cuts the DNA double helix.…”

“…Golden Gate parts that must be digested with a certain restriction endonuclease must also lack internal recognition sites for that endonuclease; the same applies to Golden Gate destination vectors, which should contain no additional recognition sites for the chosen endonuclease(s). 19,20 Importantly, it is recommended that parts of the same type, such as different ribosome binding sites, should be flanked by the same pair of fusion sites. This way, they can all be ligated in the same position (usually between a promoter and a protein coding sequence), and can be easily exchanged between laboratories, as parts made by one research group will also work for all other groups following the same rules for DNA part constructions (also called a "syntax").…”

“…For example, a DNA part beginning with a GGTCTCN ▼ AATG ▲ site will ligate with a part ending with a ▼ AATG ▲ NGAGACC site (note that NGAGACC is the reverse complement of GGTCTCN). Golden Gate parts that must be digested with a certain restriction endonuclease must also lack internal recognition sites for that endonuclease; the same applies to Golden Gate destination vectors, which should contain no additional recognition sites for the chosen endonuclease(s). , …”

“…For example, if a DNA part contains an internal BsaI site, it cannot be assembled in a BsaI-based destination vector. It is now considered best practice to remove internal sites for BbsI, BsaI, and BsmBI during part domestication, , since these enzymes are used by the most widespread assembly methods (MoClo uses BbsI and BsaI, and GoldenBraid uses BsmBI and BsaI). Still, “illegal” internal sites are sometimes encountered in older toolkits, and it is always worth checking parts for compatibility with the intended restriction enzymes.…”