Making a multi-part plasmid-V1-S2-PDF

Overview

This protocol describes how to assemble multiple-part plasmids via BsaI digestion and T7 Ligase ligation. It is intended to supplement Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology at <http://pubs.acs.org/doi/pdf/10.1021/sb500366v> (2015), as well as its supplementary documents.

Materials

• BsaI restriction enzyme.
• T4 Ligase Buffer
• T7 Ligase
• dATP
• DNA to insert

Protocol

• Make a 10 μL solution containing:
  • 20 femto moles of each plasmid
  • 5 units of BsaI, corresponding to 0.5 μL of NEB #R0535S containing 10,000 U/mL
  • 1,500 units of T7 Ligase corresponding to 0.5 μL of NEB #M0318S containing 3,000,000 U/mL.
  • 1 μL of 10X buffer for T4 Ligase with 10 mM ATP (NEB #B0202S). *Note* This buffer is T4 but the enzyme is T7; this is intentional. T7 has a preservative which prevents T7 from degrading at high temperatures. T4 is very similar to CutSmart buffer, plus it contains ATP.
  • Bring to 10 μL with DI water
• Bring the solution to the following temperatures in a thermocycler:
  • For 30 cycles
    • 42°C for 5 minutes (cutting predominantly occurs at this temperature)
    • 16°C for 5 minutes (ligation predominantly occurs at this temperature)
  • If the final product DOES NOT HAVE a BsaI dropout segment
    • Bring the solution to 42°C for 30 min hours.
    • Bring the solution to 60°C for 30 min hours. This is the ideal cutting temp. It is not reached earlier to preserve the integrity of the dissolved ATP (this is my hypothesis, the manual doesn’t explain why a non-ideal cutting temp is recommended.)
    • Bring the solution to 80°C for 10 minutes. This denatures the cutting and ligating enzymes.
  • If the final product DOES HAVE a BsaI dropout segment (OPTION 1)
    • Keep the solution at 16°C 30 min
    • Store on ice or at 4°C until the solution can be transformed into competent e. coli.
  • If the final product DOES HAVE a BsaI dropout segment (OPTION 2)
    • Bring this to the higher temperatures that would be appropriate if there was not a BsaI dropout segment.
    • After the 80°C step, bring the dATP concentration to 1 mM (assuming the dATP in T4 buffer broke down) by adding 0.1 μL of 100 mM dATP.
    • Add 0.5 μL T7 DNA ligase.
    • Leave at room temp for 1 hr (or 16°C for perhaps 30 min).
    • Proceed with transformation
• Transform this product into competent e. Coli, and select white colonies on Kanamycin.

Notes

This protocol is more involved than the one recommended in the Yeast Toolkit paper. It was designed to improve the efficiency of the transformation, and the extra steps are much better than the possibility of redoing the entire experiment. The paper’s instructions were designed for simplicity and speed. These instructions are designed to make it work.

Response from Dueber Lab (Yeast Toolkit)

Testing done in the Dueber Lab and Novome Biotechnologies found that in late 2015, NEB BsmBI and T7 ligase ceased to function well in Golden Gate assembly.

• These labs have since switched to using Thermo’s mesophilic isoschizomer Esp3I instead of BsmBI and using low-concentration T4 DNA ligase instead of T7. These have worked well.
• The cycling incubation temperature ought to be brought down to 37°C, and to restore full activity of BsaI at this temperature, add 1×BSA (bovine serum albumin). BsaI-HFv2 may not require the 1×BSA due to ligase buffer-compatibility enhancement, but it likely functions better at 37°C due to modifications changing its original thermostability.
• The addition of ~0.5–1% PEG-3350 seems to improve the number of correct assemblies ≈twofold with no impact on misassembly/parent vector counts. (make a BSA-PEG enhancer mix)
• The ligation temperature can be brought down to 16°C, though higher ligation fidelity is expected at 20°C.
• A 10–20 min initial digest ought to be considered to enhance efficiency, according to the CIDAR MoClo protocol.

References

Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology at <http://pubs.acs.org/doi/pdf/10.1021/sb500366v> (2015)