Golden Gate Cloning
Golden Gate Cloning or Golden Gate assembly[1] 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.
[4] While this technique can be used for a single insert, researchers have used Golden Gate Cloning to assemble many pieces of DNA simultaneously.
[6] 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.
[6] Golden Gate assembly uses Type IIS restriction enzymes cutting outside their recognition sequences.
[6] 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.
[6] As additional segments can be inserted into the vectors without scars within an open reading frame, Golden Gate is widely used in protein engineering.
[7] Critical to this method of assembly, the vector backbone of the destination plasmid and all the assembly fragments are flanked by Type IIS restriction enzyme recognition sites, as this subtype of restriction enzymes cut downstream from their recognition sites.
[8] Modular Cloning, or MoClo, is an assembly method introduced in 2011 by Ernst Weber et al., whereby using Type IIS restriction sites, the user can ligate at least six DNA parts together into a backbone in a one-pot reaction.
[9] 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.
[5] Level 0 modules are the base for MoClo system, where they contain genetic elements like a promoter, a 5' untranslated region (UTR), a coding sequence, and a terminator.
[5] In this process, one needs to make sure that the introduced mutation will not affect the genetic function encoded by the sequence of interest.
[5] The vector used in cloning level -1 fragments cannot contain Type IIS restriction site BpiI that is used for the following assembly step.
[10] Therefore, constructs of more than six genes need successive cloning steps, which requires end-linkers containing BsaI or BsmBI internal restriction sites and blue or purple markers.
In standard Golden Gate Cloning, the restriction sites from the previous tier construct cannot be reused.
GoldenBraid overcomes the problem of designing numerous destination vectors by having a double loop, which is the "braid," to allow binary assembly of multiple constructs.
The technology is easy to implement as a web tool is available for primer design (https://msbi.ipb-halle.de/GoldenMutagenesisWeb/) and the vectors are deposited at addgene (http://www.addgene.org/browse/article/28196591/).
