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  • Protein Expression using the K. lactis Protein Expression Kit - Transformation of K. lactis GG799 cells

    Introduction

    Introduction of the linearized expression cassette into K. lactis cells is achieved by chemical transformation using the K. lactis GG799 Competent Cells and NEB Yeast Transformation Reagent supplied with the kit.  This procedure yields approximately 1 x 104 transformants per microgram of DNA.  Transformants are selected by growth on Yeast Carbon Base (YCB) Agar Medium containing 5 mM acetamide.  YCB medium contains glucose and all nutrients needed to sustain growth of K. lactis GG799 cells except a simple nitrogen source.  Cells can utilize acetamide as a source of nitrogen only after it is broken down to ammonia by acetamidase, the product of the amdS gene present in pKLAC2.

    The following steps should be conducted using aseptic technique.  Care should be taken to ensure that pipet tips, tubes, solutions, and deionized water are sterilized prior to use.

    Protocol

    1. Thaw a tube of K. lactis GG799 Competent Cells on ice.  Add 620 μl of NEB Yeat Transformation Reagent to the cells.  Briefly shake or invert the tube until the solution is homogeneous. 

      Do not vortex.
    2. Add 1 μg of linearized pKLAC2 DNA containing the gene of interest to the cell mixture.  Briefly shake or invert the tube to mix.

      Do not vortex.  The total volume of transforming DNA should not exceed 15 μl.
    3. Incubate the mixture at 30oC for 30 minutes.
    4. Heat shock the cell mixture by incubation at 37oC for 1 hour in a water bath.
    5. Pellet cells by microcentrifugation at ~7,000 r.p.m. for 2 minutes.  Discard the supernatant.
    6. Resuspend the cell pellet in 1 ml sterile YPGlu medium.
    7. Pellet cells by microcentrifugation at ~7,000 r.p.m. for 2 minutes.  Discard the supernatant.
    8. Resuspend the cell pellet in 1 ml YPGlu medium and transfer the cell mixture to a sterile culture tube.  Incubate with shaking (250-300 r.p.m.) at 30oC for 3-4 hours.

      Incubations shorter than 3 hours are not recommended due to a decline in transformation efficiency.
    9. Transfer the cell mixture to a sterile 1.5 ml microcentrifuge tube.  Pellet the cells by microcentrifugation at ~ 7,000 r.p.m. for 2 minutes and discard the supernatant.  Resuspend the cell pellet in 1 ml sterile 1X PBS.
    10. Remove 10, 50, and 100 μl samples of the cell suspension to separate fresh sterile 1.5 ml microcentrifuge tubes, each containing 50 μl of sterile deionized water.  Mix briefly and spread the entire cell mixture from each tube onto separate YCB Agar Medium plates containing 5 mM acetamide.  Incubate plates inverted at 30oC for 3-4 days, until colonies form.

      Due to the high transformation efficiency of K. lactis GG799 Competent Cells, plating multiple dilutions of the cell mixture is necessary to ensure formation of plates with distinct single colonies.  Growth time should not exceed 5 days as small background colonies that lack an integrated expression fragment may form.  Plates containing colonies can be stored at 4oC for up to two weeks.
    11. Streak or patch 10-20 individual colonies onto freah YCB Agar Medium plates containing 5 mM acetamide.  Incubate at 30oC for 1-2 days.

      Patches of approximately 1.0 cm2 are recommended.  Plates containing patched cells may be stored at 4oC for up to 3 days prior to performing whole-cell PCR (optional steps 12 and 13).
    12. *Optional Step*

      Transformants can be tested to verify that they have correctly integrated the expression fragment. (See Identification of Properly Integrated cells).
    13. *Optional Step*

      Correctly integrated transformants can be further screened to identify cells that have integrated multiple tandem copies of the expression fragment (See Identification of Multicopy Integrants).