In order to conduct meaningful research, synthetic biologists need to create vast numbers of genetic circuits. With traditional DNA assembly methods, this process can be both time consuming and expensive. In addition, current methods allow for only one circuit variant to be made at a time. This is inefficient when specific, but unknown, parameters are needed in the circuit.

Golden Gate-Gibson (3G) is a new, hybrid method of DNA assembly that addresses these issues. In 3G assembly, many variants of multi-part circuits can be constructed in a single day with high accuracy and efficiency [1]. When implemented in our iGEM lab, 3G assembly greatly improved our productivity and enabled us to spend less time on circuit construction and more time on experiments. With a simple protocol and low costs, 3G would be an invaluable tool for the iGEM program as whole. In order to make this method of DNA assembly accessible to all iGEM teams, we created a library of compatible parts to add to the registry.

Mechanistically, 3G assembly is a hybrid of Golden-Gate and Gibson assembly. In the first stage, transcriptional units are built using Golden-Gate. In the second stage, transcriptional units are combined on to a universal backbone.

In Golden-Gate Assembly, type IIS restriction enzymes are used to cut DNA. Type IIS restriction enzymes are useful in that they cut outside their recognition sites, creating fragments of DNA with no unwanted base pair scars. We use the restriction enzyme BsaI, which recognizes specific DNA sequences (BsaI sites) and cuts outside of these sites, leaving sticky ends that can be ligated together with T4 DNA ligase.

In this stage of assembly, 3G takes advantage of the Cidar MoClo system, in which specific part types are distinguished by their sticky ends. After being cut, each type of part reveals a distinct sticky end on either side. The standard parts used in most synthetic circuits are promoters, 5’ untranslated regions, coding sequences, and terminators. Their MoClo sticky ends are shown in the image below:

The unique sticky ends allow for the parts to line up in the correct sequence before being ligated together. In this way, a full transcriptional unit can be created. To prepare for the Gibson step of 3G, unique nucleotide sequences (UNS) are attached to both ends of the transcriptional unit. The UNS on the 5’ end of the transcriptional unit must have a sticky end A so that it can anneal to the promoter’s sticky end. The UNS on the 3’ end has a sticky end E so that it can anneal to the terminator’s sticky end. These sequences serve as a landing pad for primers in the next stage of PCR. They will also be used when combining the transcriptional units on to a backbone in the final stage of 3G assembly.

There are multiple 5’ UNSs and multiple 3’ UNSs, denoted by numbers (ex: UNS 1, UNS 3, UNS 10). This allows us to combine multiple fragments in the Gibson step.