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  • K. lactis Protease Deficient Competent Cell Sampler

    Description

    Chemically competent Kluyveromyces lactis cells are suitable hosts for expressing proteins that have exhibited proteolysis in the parental GG799 background (NEB #C1001S). Proteolysis of secreted heterologous proteins in K. lactis is most often casued by aspartyl poroteases that are present in the secretory pathway. In many cases, a single aspartyl protease causes the majority of the detrimental protein degredation. The Protease Deficient Competent Cell Sampler contains a wild-type GG799 strain, as well as four protease deficient K. lactis strains, each having selectable marker-free deletion of a specific secretory pathway aspartyl protease gene. Each strain can be transformed with any linearized pKLAC-series expression vector, permitting a comparison of heterologous proein quality and yield in each background. 

    The Sampler contains one transformation each of five K.lactis strains. K. lactis GG799 is a wild-type expression strain. K. lactis YCT389 contains a selectable marker-free deletion of the KLLA0E0398g locus encoding an extracellular aspartyl protease (KlYps1p) that is homologous to cerevisiae Yps1p. K. lactis YCT390 carries a selectable marker-free deletion of the KLLA0F2208g locus encoding an extracellular aspartyl protease (Klyps7p) that is homologous to S.cervisaiae Yps7p. K. lactis strain YCT 569 carries a selectable marker-free deletion of the KLLA0D01507g locus encoding an extracellular aspartyl protease belonging to pfam00026. K. lactis strain YCT598 carries a selectable marker-free deletion of the KLLA0D15917g locus encoding an extracellular aspartyl protease that is homologous to S.cervisaiae Bar1p. 

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEB Yeast Transformation Reagent (5 ml)1X

    Properties and Usage

    Storage Temperature

    -80°C

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Sterility (K. lactis) :
      The product is tested for bacterial and/or fungal contamination by a plating assay.
    • Transformation Efficiency:
      The competent cells are tested for transformation efficiency and pass minimum release criteria. Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 μg of plasmid into a given volume of competent cells.

    Notes

    1. Due to the high transformation efficiency of K. lactis 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 colonies that lack an integrated expression fragment may form. 

      Plates containing colonies can be stored at 4°C for up to two weeks. 

      Growth of K. lactis Competent Cells carrying an integrated expression vector for protein expression should be performed in YPGal medium (1% yeast extract, 2% peptone, 2% galactose) supplemented with a final concentration of 38 mM (NH4)2SO4. In the absence of 38 mM (NH4)2SO4, YCT569, YCT389, YCT390 and YCT598 cells will grow slower than the GG799 parent strain (#C1001).
    2. Storage: Store Competent Cells at -80°C. Once Thawed, Do Not Re-freeze.

    References

    1. Colussi, P.A. et al. (2005). Appl. Environ. Microbiol. 71, 2862-2869.

    Supporting Documents

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What K. lactis protease deletion strains does NEB sell?
    1. Transformation Protocol (C1007)

    Application Notes

    Always store immediately at -80 C.  Yeast competent cells will quickly lose competency if handled improperly.