McClean: Golden Gate-Making a new part

Overview

This protocol describes how to insert a PCR product into the Yeast Toolkit entry vector via BsmBI 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.

See McClean: Designing "Yeast Toolkit" compatible primers for help making the primers to PCR amplify a Yeast Toolkit compatible part.

Materials

• PCR product which will serve as the DNA "part" to insert into an entry vector
• GeneJet gel extraction and DNA clean up
• BsmBI restriction enzyme.
• T4 Ligase Buffer
• T7 Ligase
• dATP
• Entry vector a.k.a. pYTK001 a.k.a. pMM452

Protocol

• Purify the PCR product.
  • After amplifying your part of interest by PCR, check that the PCR product is the correct size by performing gel electrophoresis on a sample of the product.
  • Remove the primers from the PCR solution with the "GeneJet gel extraction and DNA clean up kit." This kit works well and removes DNA that is <50bp. This is ideal because the oligos in the solution would otherwise compete for the restriction enzymes and could also be undesirably ligated into the entry vector during golden gate assembly.
  • Check the concentration of the purified DNA. Measuring <50ng/μL on the nanodrop is a bad sign. It can mean that there wasn't a significant amount of DNA extracted.
• Prepare a 10 μL solution for Golden Gate assembly containing:
  • 20 femto moles of the entry vector pMM452 a.k.a. pYTK001.
  • 20 femto moles of the DNA insert which has BsmBI cut sites
  • 10 units of BsmBI, corresponding to 1 μL of NEB #R0580S 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 2 minutes (cutting predominantly occurs at this temperature)
    • 16°C for 5 minutes (ligation predominantly occurs at this temperature)
  • 55°C for 1 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.)
  • 80°C for 10 minutes. This denatures the cutting and ligating enzymes.
• Transform this product into competent e. Coli, and select white colonies on Chloramphenicol. Innoculate the competent e. Coli with 0.5μL of the Golden Gate solution (~0.5 fMol of DNA) dissolved in 9.5μL DI water.

Notes

This protocol is very similar to the protocol recommended in the Yeast Toolkit paper except that it recommends a 2 minute cutting period at 42 °C instead of a 2-minute cutting period, and it recommends a 1-hour cutting period at 55°C to drastically reduce the number of undigested backbone plasmids. The 55°C step can be skipped to save time at the expense of having more undigested pMM452 in the final transformation mix (and thus more undesirable GFP-expressing e. Coli after selection on LB+Chloramphenicol). Parts of this protocol may seem non-ideal to make sense: we recommend the wrong cutting buffer for BsmBI, non-ideal temperatures, and excessive amounts of enzymes. The overarching reason is that we are attempting to both cut and ligate at in the same solution, and so we need to deviate from the optimal reaction conditions of the individual components and compensate for the reduced efficiencies with excess enzymes.

• Cameron J. Stewart 13:46, 15 September 2016 (EDT):

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)

Contact

• Cameron J. Stewart 13:46, 15 September 2016 (EDT):
• Megan N McClean 14:01, 20 July 2011 (EDT)

or instead, discuss this protocol.