Golden Gate Cloning

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. 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. 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 thermal cycler 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. 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, an 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 remain 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. Although Golden Gate Cloning speeds up multisegment cloning, careful design of donor and recipient plasmids is required. In each cloning step, Golden Gate Cloning can assemble up to nine fragments and only requires homology in type II restriction enzyme sites so that the DNA fragments can be ligated seamlessly. After the fragments are ligated, the product will not have the original type IIS restriction site and will not be redigested in ligation reaction afterwards. Meanwhile, the original restriction sites, which are not ligated, can be redigested so that they can add more fragments into the plasmid. If the DNA fragments are well-designed to be compatible to one another, they can be ligated in a linear order in one step. 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 and 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 reading frames, and terminators. Then, second-tier Golden Gate assembly combine several constructs made in first-tier assembly to make a multigene construct. To achieve second-tier assembly, modular cloning(MoClo) system and GoldenBraid2.0 standard are used. MoClo utilizes a parallel approach, where all constructs from tier-one(level 0 modules) 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. LacZ is a common screening cassette, where it is replaced by the multigene construct on the destination vector. Each tier-one construct and the vector have different overhangs on them yet complementary to the overhang of the next segment, and this determines the layout of the final multigene construct. Golden Gate cloning usually starts with level 0 modules. However, if the level 0 module is too large, cloning will start from level -1 fragments, which have to be sequenced, to help cloning the large construct. If starting from level -1 fragments, the level 0 modules do not need to be sequenced again, whereas if starting from level 0 modules, the modules must be sequenced.