Home Protocols Protein Expression using the K. lactis Protein Expression Kit - Identification of properly integrated cells

Protein Expression using the K. lactis Protein Expression Kit - Identification of properly integrated cells

Overview

Transformants in which the expression cassette has correctly integrated into the K. lactis genome can be identified by PCR using supplied Integration Primers 1 and 2 to amplify a 2.65 kb product.  To facilitate simultaneous screening of many transformants, PCR using freshly grown cells as a course of template chromosomal DNA is recommended.

Protocol

  1. For each transformant patched and grown on YCB Agar Medium plates containing 5 mM acetamide, harvest cells from an area approximately 1 mm2 by scraping with a pipette tip and resuspend the cells in 100 μl of 0.2 M LiAc containing 1% w/v SDS). 
  2. Incubate at 70°C for 15 minutes then add 300 μl 100% ethanol and vortex to mix.
  3. Centrifuge the cells at 15,000 x g for 3 minutes at room temperature.
  4. Carefully remove the supernatant and air dry the pellet for 10 minutes.
  5. Resuspend the pellet in 50 μl TE (10 mM Tris-HCl, pH 7.5 containing 1 mM EDTA).
  6. Centrifuge to remove any insoluble material for 30 seconds at 13,000 x g.
  7. Transfer the supernatant to a fresh tube and keep on ice. Use 1 μl as a template for a 50 μl PCR reaction as follows: 

    3.2 μl 10X Integration Primer 1 (7.8 μM stock)
    3.2 μl 10X Integration Primer 2 (7.8 μM stock)
    25 μl Q5® High-Fidelity 2X Master Mix (NEB #M0492)
    1.0 μl LiAc/SDS treated supernatant from Step 7 above
    17.6 μl deionized water
    ----------------------------------------------------------------------------------
    50 μl final reaction volume

  8. Thermocycling should consist of an initial denaturation at 98°C for30 seconds followed by 30 rounds (98°C for 10 seconds, 60°C for 30 seconds and 72°C for 75 seconds) and a final extension at 72°C for 2 min. 
  9. Analyze 10 μl of each amplification on a 1% agarose gel.
    Integration of the expression fragment at the LAC4 locus in the K. lactis genome will result in amplification of a 2.65 kb product.
  10. Test strains harboring a properly integrated expression fragment for secretion of the protein of interest.