Golden Gate cloning or Golden Gate assembly is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4 DNA ligase. This assembly is performed in vitro. Most commonly used Type IIs enzymes include BsaI, BsmBI and BbsI.
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Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences. In practice, this means that Golden Gate cloning is typically scarless. Additionally, because the final product does not have a Type II restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible.
A typical thermocycler protocol oscillates between 37 °C (optimal for restriction enzymes) and 16 °C (optimal for ligases) many times. While this technique can be used for a single insert, researchers have used Golden Gate cloning to assemble many pieces of DNA simultaneously.
Quasi-Scarless
Scar sequences are common in multiple segment DNA assembly. In the multisegment assembly method Gateway, segments are added into the donor with additional att sequences, which overlap in those added segments, and this results in the segments separated by the att sequences. In BioBrick assembly, a eight-nucleotide scar sequence, which codes for a tyrosine and a stop codon, is left between every segment added into the plasmid.
Golden Gate assembly uses type II restriction enzymes cutting outside their recognition sequences. Also, the same type II restriction enzyme can generate copious different overhangs on the inserts and the vector, for instance, BsaI creates 256 four-basepair overhangs. If the overhangs are carefully designed, the segments are ligated without scar sequences between them, and the final construct can be quasi-scarless, where the restriction enzyme sites are remained on both sides of the insert. As additional segments can be inserted into the vectors without scars within an open reading frame, Golden Gate is widely used in protein engineering.
Cloning Standards
Restriction enzyme DNA assembly has cloning standards to minimize the change in cloning efficiency and the function of the plasmid, which can be caused by compatibility of the restriction sites on the insert ant those on the vector.
Golden Gate assembly's cloning standards have two tiers. First-tier Golden Gate assembly constructs the single-gene construct by adding in genetic elements such as promoter, open read frames and terminators.Then, second-tier Golden Gate assembly combine several constructs made in first-tier assembly to make a mutigene construct. To achieve second-tier assembly, modular cloning(MoClo) system and GoldenBraid2.0 standard are used. On one hand, MoClo utilizes a parallel approach, where all constructs from tier-one have restriction sites for BpiI on both sides of the inserts. The vector(also known as "destination vector"), where genes will be added, has an outward-facing BsaI restriction site with a drop-out screening cassette. Each tier-one construct and the vector have different overhangs on them yet complimentary to the overhang of the next segment, and this determines the layout of the final multigene construct. On the other hand, GoldenBraid uses a pairwise approach, where two tier-one constructs with BsmBI restriction sites pair up and are added into a vector with BsaI sites, which are farther from both inserted genes. Then, the constructs with two genes and BsaI sites are added into the destination vector with BsaI sites, and the final multigene construct with have BsmBI restriction sites flanking around the genes added.