The Golden Gate assembly protocol was defined by Engler et al. 2008 to define a DNA assembly method that would give a final construct without a scar sequence, while also lacking the original restriction sites. This allows the protein to be expressed without containing unwanted protein sequences which could negatively affect protein folding or expression. By using the BsaI restriction enzyme that produces a 4 base pair overhang, up to 240 unique, non-palindromic sequences can be used for assembly.
Plasmid design and assembly
In Golden Gate cloning, each DNA fragment to be assembled is placed in a plasmid, flanked by inward facing BsaI restriction sites containing the programmed overhang sequences. For each DNA fragment, the 3 overhang sequence is complementary to the 5 overhang of the next downstream DNA fragment. For the first fragment, the 5 overhang is complementary to the 5 overhang of the destination plasmid, while the 3 overhang of the final fragment is complementary to the 3 overhang of the destination plasmid. Such a design allows for all DNA fragments to be assembled in a one-pot reaction (where all reactants are mixed together), with all fragments arranged in the correct sequence. Successfully assembled constructs are selected by detecting the loss of function of a screening cassette that was originally in the destination plasmid.
MoClo and Golden Braid
The original Golden Gate Assembly only allows for a single construct to be made in the destination vector . To enable this construct to be used in a subsequent reaction as an entry vector, the MoClo and Golden Braid standards were designed.
The MoClo standard involves defining multiple tiers of DNA assembly:
* Tier 1: Tier 1 assembly is the standard Golden Gate assembly, and genes are assembled from their components parts (DNA parts coding for genetic  elements like UTRs, promoters, ribosome binding sites or terminator sequences). Flanking the insertion site of the tier 1 destination vectors are a pair of inward cutting BpiI restriction sites. This allows these plasmids to be used as entry vectors for tier two destination vectors.
* Tier 2: Tier 2 assembly involves further assembling the genes assembled in tier 1 assembly into multi-gene constructs. If there is a need for further, higher tier assembly, inward cutting BsaI restriction sites can be added to flank the insertion sites. These vectors can then be used as entry vectors for higher tier constructs.
Each assembly tier alternates the use of BsaI and BpiI restriction sites to minimise the number of forbidden sites, and sequential assembly for each tier is achieved by following the Golden Gate plasmid design. Overall, the MoClo standard allows for the assembly of a construct that contains multiple transcription units, all assembled from different DNA parts, by a series of one-pot Golden Gate reactions. However, one drawback of the MoClo standard is that it requires the use of dummy parts with no biological function, if the final construct requires less than four component parts. The Golden Braid standard on the other hand introduced a pairwise Golden Gate assembly standard.
The Golden Braid standard uses the same tiered assembly as MoClo, but each tier only involves the assembly of two DNA fragments, i.e. a pairwise approach. Hence in each tier, pairs of genes are cloned into a destination fragment in the desired sequence, and these are subsequently assembled two at a time in successive tiers. Like MoClo, the Golden Braid standard alternates the BsaI and BpiI restriction enzymes between each tier.
The development of the Golden Gate assembly methods and its variants has allowed researchers to design tool-kits to speed up the synthetic biology workflow. For example, EcoFlex was developed as a toolkit for E. Coli that uses the MoClo standard for its DNA parts, while a similar toolkit has also been developed for engineering the Chlamydomonas reinhardtii microalgae.