Team:William and Mary/3GProtocols

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3G Assembly Protocols

Overview
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.
An overview of 3G Assembly.
Design:
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.
In the next step, transcriptional units are amplified with PCR. The forward primer will anneal to the 5’ UNS and the reverse primer will anneal to the 3’ UNS. The PCR products are then gel extracted.
In the Gibson stage, transcriptional units are attached to each other and to a backbone. The backbone must have UNS 1 and UNS 10 inside the BioBrick Prefix and Suffix in order for the transcriptional units to attach. As mentioned earlier, the UNSs on each transcriptional unit determine the order they end up in, as shown below. Up to six transcriptional units can be put on a backbone in 3G assembly.
Transcriptional units are either supplied from the iGEM registry or can be designed. They may need to be cloned on a backbone in order to increase volume. Oligo adapters are then added.
Protocol:

Preparation of 50 nM Oligo Adapters (UNS)

Single stranded lyophilized oligos (see table below) are resuspending in IDT duplex buffer to a concentration of 100 uM. Top and bottom oligos are combined in a 1:1 molar ratio in duplex buffer to a final concentration of 1µM and then annealed by heating to 98C for 1 minute and then cool to room temperature on the benchtop. 50 nM working stocks are created by a 1:20 dilution of the 1µM stock in NFW.

Preparation of UNS1/10 iGEM Backbones

The UNS1/10 iGEM backbones can be prepared by a standard PCR. To a .2 mL tube, add

-1.25 µL primer BBa_K2680513
-1.25 µL primer BBa_K2680514
- 9 µl NFW, 1 ul of a miniprep of either pSB1C3 or pSB3K3
- 12.5 µl of Q5 2x Master Mix.

Program the thermocycler with standard PCR cycles with a 55 C annealing temperature and a 1 minute extension time for pSB1C3, or a 1 minute 30 second extension time for pSB3K3. Gel extract the PCR product.

Preparation of 30 nM 3G parts

Quantify the concentration of the miniprepped 3G part with a nanodrop, convert to molarity, then dilute to 30 nM in nuclease free water.
References
[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