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  • Protein Expression using the K. lactis Protein Expression Kit - Identification of Multi-copy Integrants

    Overview

    It is possible for up to 10 copies of the expression casette to tandemly insert into the genome during transformation.  Strains harboring multiple integrations often produce more secreted protein.  An advantage of selection for K. lactis transformants on YCB Agar Medium containing acetamide is that it enriches for cells harboring multiple tandem integrations.  Multiply integrated cells can be identified using whole-cell PCR using Integration Primers 2 and 3.

    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 25 μl of 1 M sorbitol containing 2 mg/ml Lyticase (Sigma #L-2524). Mix by vortexing. Incubate at 30oC for 1 hour.
    2. Lyse the Lyticase-treated cells in a thermocycler at 98oC for 10 minutes.
    3. Mix
      25 μl - Lyticase treated cells
      10 μl - 10X Integration Primer 2
      10 μl - 10X Integration Primer 3
      10 μl - 2mM dNTPs (NEB #N0447, 10 mM stock)
      10 μl - 10X ThermPol Buffer (NEB #B9004)
      1 μl - Taq DNA Polymerase (NEB # M0267 or NEB # M0273)
      34 μl - deionized water
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      100 μl - final reaction volume
    4. Thermocycling should consist of 30 rounds (94oC for 30 seconds, 50oC for 30 seconds and 72oC for 2 minutes), followed by incubation at 72oC for 10 minutes.
    5. Analyze 10 μl of each amplification reaction on a 1% agarose gel.

      Cells harboring multiple tandem integrations of the expression fragment at the LAC4 locus in the K. lactis genome will result in amplification of a 2.3 kb product.
    6. Test strains harboring multiple copies of the expression fragment for secretions of the protein of interest (See Growth of strains for detection of secreted protein).