Golden Gate cloning 1 is a rapidly developing technology that utilises Type IIS Restriction endonucleases (Type IIS RENs) and allows rapid and simple construction of multi-gene constructs - an otherwise time-consuming process. Unlike the more commonly-used Type II RENs, Type IIS RENs cleave downstream of their recognition sequence, meaning that the recognition sequence is removed from the assembly product. Due to the removal of the recognition site from the assembly product, the reaction is irreversible meaning that the reaction will always tend towards the desired product. This allows for multi part assembly in a single digestion-ligation (dig-lig) reaction dramatically speeding up the process and efficiency of cloning whilst producing a recombinant plasmid that is free from unwanted sequences. The Golden Gate Modular Cloning (MoClo) assembly standard 2-4, defines a fixed set of sequences to the overhangs produced when parts are released from their vector by the TypeIIS enzyme. The sequence defines the position the part will occupy within the final construct. In order to achieve this, the part must be flanked by standardised, predefined overhangs that fit the MoClo standard. The advantage of using any standardized assembly system is that, as well as dramatically speeding up the cloning process, it allows us to share our work with other laboratories and iGEM teams. The Green Canary project has utilised Golden gate cloning and the MoClo standard to assemble all the constructs required for the project from previously synthesised level 0 modules. Specifically, the enzymes used are BsaI to assemble level 1 constructs from level zero parts and Bpi1 for assembly of level two constructs from level one constructs. The recognition sites for BsaI and BpiI are not palindromic and are therefore directional, being either convergent or divergent in orientation.

The recognition and cleavage sites of the Type IIS RENs utilised are as follows:
BpiI (BbsI) - GAAGACNN↓NNNN
BsaI - GGTCTCN↓NNNN
Where N is any nucleotide

The cleavage sites, which will remain in the assembly product and are usually 4bp in length, can be composed of any sequence (as shown above- N is any Nucleotide). These overhangs are often designed to be a section of the part itself, meaning that even the remaining cleavage sites leave no scar. The accuracy of ligation is dependent on the length of the overhangs; therefore 4bp overhangs are preferred. There is a risk of mis-ligation by similar overhangs and therefore it is ideal for the overhangs to be at least 50% different in order to efficiently assemble multiple fragments in one reaction. All the 4bp overhangs defined in the MoCLo assembly standard conform to this rule.

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Figure 1: Level 0 parts consist of sequences with standard overhangs that have been cloned into a level 0 acceptor using the Type IIS REN BpiI. The level 0 acceptor has two inverted BpiI sites and two inverted BsaI sites. The BpiI sites are convergent on each insert and divergent on the level 0 acceptor, therefore will be removed by the process of cloning and will not exist in the assembled level 0 part. The result of the Golden Gate cloning reaction is a level 0 part that is flanked by inverted BsaI sites allowing release of the part using BsI in subsequent Golden Gate reactions.

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Figure 2: Level 0 parts can be combined in a Golden Gate dig-lig reaction with the appropriate level one acceptor to create a level one construct that will usually consist of a complete transcriptional unit. The BsaI sites are convergent on each level 0 part and divergent on the level 1 acceptor, therefore will be removed by the process of cloning and will not exist in the assembled level 1 construct. The assembly product has no restriction sites for Bsa1 but is flanked by inverted, convergently orientated BpiI sites. The Level one acceptor must be chosen according to which position the construct will occupy in the prospective level two construct (see Figure 4).

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Figure 3: Level two cloning allows assembly of multiple, whole transcriptional units into one construct in a single step. The Bpi1I sites are convergent on each level 1 part and divergent on the level 2 acceptor, therefore will be removed by the process of cloning. The product of this assembly is multiple transcriptional units within a level 2 acceptor, creating a level two, multigene construct in which neither Bsa1 nor Bpi1 restriction sites remain.

Multiple level one constructs can be assembled into a multi gene (level 2) construct whilst still maintaining high efficiency. “Endlinkers” join the compatible overhang of the final construct to the GGGA of the level 2 acceptor, according to the number of genes in the assembly as shown below. “Dummies “can also be used to substitute for any position.

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Figure 4: Design of a level 2 cloning reaction showing the Endlinker to be used according to the number of genes within the construct.

The antibiotic resistance of the acceptors is different for each level, allowing selection of the new, desired construct. This is also standardised: level 1 acceptors are carbenicillin/ ampicillin resistant and level 0 and 2 acceptors are spectinomycin resistant.

Domestication
Domestication of the parts is required before use in Golden Gate cloning reactions. This involves removing internal recognition sites already in the part to prevent internal overhangs from annealing to desired overhangs, creating incorrect assemblies.

Whilst BioBrick assembly is a powerful tool in Synthetic Biology due to the large quantity of standardised parts available through the Registry, genetic assembly of complex multigene constructs using conventional cloning is time consuming as only two parts can be assembled in each reaction. Golden Gate technology is a reliable alternative, which can dramatically speed up the assembly of larger constructs as multiple modules can be assembled in a single dig-lig reaction. Development of the MoClo standard allows laboratories to access the increasing number of available parts, allowing research collaboration.