Welcome to Chang Liu Team
Trees cell wall formation and wall signal Group
Golden Gate Assembly

Golden Gate Assembly

Background and Design Golden Gate cloning or Golden Gate assembly is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIs restriction enzymes and T4/T7 DNA ligase. This assembly is performed in vitro. Most commonly used Type IIs enzymes include BsaI, BsmBI and BbsI. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and therefore can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences.

Supplies

  • 10× T4 DNA ligase buffer (NEB: M0202T)
  • Why T4 DNA ligase buffer with T7 DNA ligase? T7 buffer contains PEG, which inhibits electroporation.
  • T7 DNA ligase (NEB: M0318S) ○ Why T7 DNA ligase instead of T4 DNA ligase? T7 does not ligate blunt ended DNA, yielding less off-target ligation. • Restriction endonuclease BsaI (NEB: R0535) or BsmBI (NEB: R0580S) • Competent cells • SOC and liquid media • Selective plates

Protocol Use the Calculator Spreadsheet (linked at the bottom of the page) for planning your reactions! 1. Mix 20 fmol (1 nM final concentration) backbone and 40 fmol each insert of your DNA segments together. The volume of this mixture must be 16 µL. 2. Add water to a final volume of 16 µl 3. Add 2 µL of 10× T4 DNA ligase buffer. Mix by vortexing. 4. Add 1 µL of BsaI or BsmBI and 1 µL of T7 DNA ligase. Mix by gently pipetting. 5. Incubate the reaction for 30 temperature cycles (42°C for 5 min and then 16°C for 5 min), followed by a final 10 min incubation at 55°C. Alternate cycling protocols are described below, and may show better assemblies in some situations. 6. Use 2 µL of this assembly reaction for electroporation or 4 µL for transforming chemically competent cells <- recommend change to 1uL.

Tips

  • Inserts should be screened for the presence of internal BsaI/BsmBI sites before they are used! You may need to edit these out.

• The best option for Golden Gate assembly of modules involving inserts < 250 bp or > 3kb, or inserts containing repetitive elements that might accumulate errors during PCR amplification, is to clone these into the entry vector and use that for the assembly. For other modules, PCR amplicons can usually be used in place of parts cloned into the entry vector to save time. • A 2:1 insert:destination plasmid ratio is recommended, although the Golden Gate Assembly process is robust enough that 1:1 ratios also can be used. For PCR amplicons, the amount of each insert to be added can be calculated by molar calculations or relative length calculations. • For the assembly of the transcriptional unit a 3:1 ratio (insert to backbone) seemed to be better at reducing background • Single insert cloning is more efficient than multiple insert cloning. Assembly efficiency decreases as the number of fragments increases. The presence of repetitive sequences in an insert will also decrease efficiency. For inserts < 250 bp or > 3 kbp, pre-cloning these inserts into the entry vector will increase efficiency. • The normal restrictions on overall plasmid size to allow transformation and stable maintenance in E. coli apply to Golden Gate assemblies. Efficiencies are highest when the product plasmid is < 12 kb. Larger assemblies can be made but may require larger numbers of colonies to be screened for the correct full length assembled products. • Cycling assemblies using 5-10 min temperature steps work well for larger scale assemblies (>10 inserts) and for any assembly for which maximal assembly yields and transformation levels are desired.

Troubleshooting

• No colonies on selective plate - This can be caused by many issues. Check the following: ○ Are your competent cells very competent? Low efficiency competent cells cause a working assembly to appear broken. Electrocompetent E. coli should be of ~1e4cfu/ng puc18 efficiency. ○ Are you plating the entire recovery mixture? Even with high efficiency cells, the number of successfully transformed cells can be quite low with multipart assembly (6+ parts). Spin down and plate the entire transformation on your selective media. ○ Are your parts correct? Over time plasmids can mutate, be mislabelled, or suffer from DNase contamination. Sequence all of your parts, and check for degradation. ○ How much of your assembly did you transform? Use 2µL to transform electrocompetent cells, and more if using chemically competent cells. • High number of fluorescent colonies - When using a fluorescent dropout vector (pYTK001, pYTK095, pBTK403, pBTK402), the majority of your colonies should be non-fluorescent. A high number of fluorescent colonies indicates these dropouts are not being cut efficiency, and is often caused by the following: ○ Expired BsaI or BsmBI enzymes. As with other enzymes, BsaI and BsmBI can lose their potency over time or with extensive handling at room temperature. Perform a diagnostic digest, or order and use fresh BsaI / BsmBI. Alternate Thermocycler Conditions • For the assembly of the transcriptional unit using BsaI, I found that the following protocol worked more efficiently: ○ Step 1: 37°C for 5 minutes ○ Step 2: 16°C for 5 minutes ○ (repeat steps 1 & 2 30x) ○ Step 3: 37°C for 10 minutes • If you're not using a pre-screened parts kit (YTK, MoClo, etc), occasionally your PCR amplified parts will contain internal BsaI or BsmBI sites. In these situations, it's critical you end the assembly on a ligation step. Even with 2 internal BsaI sites, I've effectively used the following two-step, non-cycling protocol: ○ For these reactions, add only BsaI initially (total volume 19µL) ○ Step 1: 37°C for 30 minutes (optimal cutting temperature for BsaI) ○ Step 2: 65°C for 20 minutes (heat inactivation of BsaI) ○ Step 3: Add 1uL T7 ligase to each reaction. ○ Step 4: 25°C for 30 minutes (optimal ligation temperature for T7 ligase) ○ Step 5: 65°C for 20 minutes (heat inactivation of T7 ligase) ○ Step 6: Electroporate 1-2µL reaction into appropriate electrocomp cells (larger volumes inhibit transformation). If using chemically competent or Z-comp cells, you can add arbitrarily large amounts of reaction. References 1. Engler C, Kandzia R, Marillonnet S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. Link 2. Lee ME, DeLoache WC, Cervantes B, Dueber JE. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4:975–986. Link 3. "Golden Gate Assembly". New England Biolabs. Retrieved 8 June 2015. Link