3G assembly is a new and cutting edge hybrid method of DNA assembly first described in 2018 by A. D. Halleran, A. Swaminathan and R. M, Murray [1] combines Golden Gate and Gibson to allow for modular assembly of multi-part circuits in a single day. In this method circuits are composed of modular transcriptional units which are constructed using Golden Gate Assembly. These transcriptional units are amplified with PCR, purified via gel extraction, then combined into a circuit using Gibson Assembly.

A circuit is composed of transcriptional units. Each transcriptional unit consists of a promoter, a 5’ untranslated region (UTR), a coding sequence (CDS), and a terminator in that order. Each of these four parts have distinct sticky ends according to the Cidar Modular Cloning System, which can be seen in the table below:

During the Golden Gate stage, the restriction enzyme BsaI cuts inside of its recognition site to reveal each part’s sticky ends so that they can be ligated together in the correct order. In addition, unique nucleotide sequences (UNS) are attached at the 5’ and 3’ end of the transcriptional unit using oligo adapters. The UNS on the 5’ end must have an A sticky end so that it can attach to the promoter, and the UNS on the 3’ end must have an E sticky end to attach to the terminator.

There are a variety of UNS sequences that can be used. In order for the transcriptional units to attach to the backbone in the Gibson stage, the first unit to go on the circuit must begin with UNS 1, and the last unit on the circuit must end with UNS 10. When creating transcriptional units that are intended to end up on a circuit together, it is important to ensure that their UNSs overlap. For instance, if the first unit is flanked by UNS 1 and UNS 3, the second must be flanked with UNS 3 and UNS 10.

[1] Single Day Construction of Multigene Circuits with 3G Assembly Andrew D. Halleran, Anandh Swaminathan, and Richard M. Murray. ACS Synthetic Biology 2018 7 (5), 1477-1480. DOI: 10.1021/acssynbio.8b00060