Murray:3G Assembly

Curators

Andrew Halleran, California Institute of Technology, 1200 E California Blvd, Pasadena, CA 91125. email: ahalleran@caltech.edu

Overview

This protocol describes how to make multiple-expression unit plasmids using Golden Gate and Gibson (3G) assembly. In 3G assembly, a golden gate assembly is used to assemble individual expression units from a library of part plasmids. Each assembly is flaked by universal nucleotide sequences (UNSs), which allow them to be easily Gibson-assembled together into a single multi-gene construct.

This protocol is based on Halleran, A. D., Swaminathan, A., & Murray, R. M. (2018). Single Day Construction of Multigene Circuits with 3G Assembly. ACS Synthetic Biology. https://doi.org/10.1021/acssynbio.8b00060.

Materials

Reagents

• Golden Gate Master Mix (BsaI-HFv2)
• T4 ligase buffer (comes with the above kit)
• Golden gate promoter, UTR, CDS, and terminator parts (either plasmid or linear DNA)
• 5' and 3' UNS adapters (made from annealed oligos; sequences can be found here: https://www.biorxiv.org/content/biorxiv/suppl/2018/02/10/260851.DC1/260851-1.pdf).
  • Combine forward and reverse primers for the adapters at 5 µM in IDT duplex buffer (100 mM Potassium Acetate; 30 mM HEPES, pH 7.5).
  • Heat the mix in a thermal cycler to 94°C for 2 minutes and cool by 1°C every minute until room temperature.
  • Dilute to 50 nM in IDTE or nuclease-free water.
• UNS amplification primers (forward and reverse 40bp primers of sequences listed here.
• A linearized destination vector containing flanking UNS sequences (typically UNS1 and UNSX). These can be easily generated from an existing backbone via overhang PCR.
• PCR master mix, or appropriate enzymes and buffer (recommend Q5 High-Fidelity 2x Master Mix).
• Gel Extraction Kit and MinElute columns.
• Gibson master mix, or appropriate enzymes. We recommend HiFi DNA Assembly Master Mix.
• Competent cells.

Equipment

• Thermal cycler

Procedure

The overall workflow for 3G assembly is:

• Golden gate assemble single-gene constructs into UNS adapters.
• PCR amplify golden gate assemblies.
• Gel extract PCRs.
• Gibson-assemble gel-extracted golden gate assemblies into a pre-made vector.
• Transform Gibson assemblies.

Before the golden gate reaction, you will need to prepare UNS adapters. Adapters are made from annealed and diluted primers. Adapter sequences can be found in this paper. Oligos are annealed following protocol and subsequently diluted to 50nM in 1x IDTE.

Golden Gate Reaction

We recommend using NEB's Golden Gate Assembly Mix with BsaI-HFv2 (NEB E1601).

In nuclease-free water, mix:

• 3 nM each part plasmid
• 1.5 nM vector plasmid
• 1x enzyme mix
• 1x buffer

For a 10 μL golden gate reaction using 30 nM stock part 50 nM stock adapters, this will be:

• 1 µL promoter part
• 1 µL UTR part
• 1 µL CDS part
• 1 µL terminator part
• 0.5 µL forward UNS adapter
• 0.5 µL reverse UNS adapter
• 0.5 µL NEB Golden Gate Master Mix
• 1 µL T4 ligase buffer
• 3.5 µL nuclease-free water

Incubate this reaction as follows:

Thermocycler Protocol

Num	Temp (°C)	Duration (s)
1	37	60
2	16	60
3	Goto 1	30-100x
4	55	300
5	4	inf

50 cycles is a typical recommended reaction time (∼2 hours). If you are pressed for time, 30 cycles is usually sufficient to produce plenty of colonies.If you are not under any time constraints, or you have a particularly difficult assembly (e.g., one with very large parts), you can increase your reaction efficiency by running more cycles, up to about 100x (∼4.5 hours).

PCR

We use NEB Q5 2x High-Fidelity Master Mix for our 3G PCRs. If you use a different enzyme, adjust your protocol accordingly.

50uL PCRs are required for high yields following gel extraction. In the 50uL PCR use, for example:

• 25 µL NEB Q5 HF 2x MM
• 18 µL nuclease free water
• 2.5 µL 40bp UNS forward primer @40µM
• 2.5 µL 40bp UNS reverse primer @40µM

Incubate this reaction according to your enzyme’s protocol. We use 64°C melt temperature and 27 cycles, i.e.:

Thermocycler Protocol

Num	Temp (°C)	Duration (s)
1	98	30
2	98	10
3	64	20
4	72	30/kb
5	Go to 2	27x
5	4	inf

Gel Extraction

PCR products should be run on a gel and gel extracted for highest efficiency assemblies. The following protocol has been optimized for purity and DNA concentration:

• To your gel fragment, add 900 µL buffer QG
• Incubate at 50°C for 10 minutes, vortexing a few times throughout
• Add 300 µL isopropanol, pipet to mix, and spin through a Qiagen MinElute column (https://www.qiagen.com/us/shop/sample-technologies/dna/genomic-dna/minelute-gel-extraction-kit/#orderinginformation)
• Discard flow through
• Spin through remaining isopropanol / QG mix
• Discard flow through
• Add 700 µL buffer PE to the column, invert the tube to wash QG from the sides and lid
• Incubate at room temperature for five minutes
• Spin and discard flow through
• Add 700 µL buffer PE to the column, invert the tube to wash QG from the sides and lid
• Spin and discard flow through
• Add 700 µL buffer PE to the column, invert the tube to wash QG from the sides and lid
• Spin and discard flow through
• Spin for 1 minute on max speed to dry column
• Transfer the column to a fresh tube
• Add 15 µL pre-warmed elution buffer to the column
• Incubate at room temperature for one minute
• Spin for 1 minute on max speed to elute
• (optional) for increased (~15%) yield, run the eluate through the column again.
• Nanodrop DNA

Gibson Assembly

For a 5uL Gibson assembly reaction we have found that including at least 1 µL of water increases the number of colonies we get. Thus, our standard protocol for Gibson assembly reactions is as follows:

• 2.5 µL 2x NEB HiFi master mix
• 1 µL Nuclease-free water
• Equimolar concentration of fragments to assemble up to 1.5 µL total volume

• Incubate at 50°C for 1 hour

Transformation

Transformation protocols vary from strain to strain. Regardless of cell strain, use 2uL of my Gibson assembly mix per 50uL of chemically competent cells.