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] which combines Golden Gate and Gibson assembly techniques 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 image 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.

Single stranded lyophilized oligos (see table in Additional Information) 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.

The UNS1/10 iGEM backbones can be prepared by a standard PCR. To a 0.2 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.

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

To a 0.2 mL tube, add the following:

• 0.5 µL promoter 30 nM miniprep stock
• 0.5 µL 5’ UTR 30 nM miniprep stock
• 0.5 µL CDS 30 nM miniprep stock
• 0.5 µL terminator 30 nM miniprep stock
• 0.5 µL 50 nM left UNS adapter
• 0.5 uL 50 nM right UNS adapter

In addition, either add:

• 0.5 µL 10x DNA ligase buffer
• 0.25 µL BsaI
• 0.25 µL T4 DNA Ligase
• 1 µL NFW

OR

• 0.5 µL Golden Gate Buffer
• 0.25 µL NEB Golden Gate Master Mix
• 1.25 µL NFW

Note: if performing multiple Golden Gate reactions, we recommend making a master mix with a slight upscale, then add 2 µL of the master mix to reaction.

Cycle the reaction in the thermocycler. For the Golden Gate Master Mix perform 30 cycles of incubating at 37 for 1 minute then incubating at 16 C for one minute. For the T4 ligase and BsaI, perform 9 cycles at 37C for 3 minutes followed by incubating at 16 C for 4 minutes. For both options, hold at 37 C for five minutes, then hold at 4 C. For information on creating multiple circuit variants at once, see parallel 3G Assembly.

Amplify the product with a 50 uL PCR as according to the PCR protocol. Add:

• 1.5 µL Golden Gate assembly product
• 18.5 µL NFW
• 2.5 µL of the appropriate 10uM UNS_foward Primer (that will anneal to the UNS on the 5’ end of your transcriptional unit)
• 2.5 µL 10 uM of the appropriate UNS_reverse primer (that will anneal to the UNS on the 3’ end of your transcriptional unit)
• 25 µL 2x Hot Start Master Mix

Incubate reactions using the standard 2x Q5 Hot Start Master Mix protocol with 27 cycles, using 30 seconds per kilobase pair, and an annealing temperature of 64 C. Conduct a gel extraction on the PCR product. (Note that 3G often produces off target bands as well as an extremely bright on target band. This is normal, and is why gel extraction rather than PCR purification is recommended).

Perform a 5 uL Gibson assembly using the purified transcriptional units and vectors. Add to a .2 mL tube:

• .015 pmolar each fragment
• .015 pmolar desired backbone
• Add NFW to a total volume of 2.5 µL.
• 2.5 ul 2X HiFi DNA Assembly master mix

Ensure the total fragment volume of the PCR product and backbone does not exceed 2.5 µl. Place tube in a thermocycler, run for 50 C for an hour, and hold at 4 C. Proceed to a transformation fairly quickly as Gibson products are only stable for a few hours.

To design a new 3G part, use Gene Block to design a new sequence of double stranded DNA. Include a 40 bp pad sequence on either end, along with a BsaI site and sticky ends appropriate for the part type (for more information on pad sequences, see 3G Assembly). This allows the new part to be cloned onto a William and Mary pad backbone.

In general, when 3G assembly fails, it will fail at the gel extraction phase. If correctly sized DNA is obtained from the gel, the Gibson reaction works very well. For circuits that fail to amplify at the PCR step, we recommend running an increased number of golden-gate cycles (100 or 30 for NEB and BsaI respectively). Additionally, use of longer (40 bp), full length UNS amplification primers may be helpful for amplification.