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DNA-Mediated Transformation

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Pichia Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 389))

Abstract

Several methods for DNA-mediated transformation of Pichia pastoris have been developed which vary in type of DNA that is transformable (e.g., linear versus circular) efficiency, cost, and labor and each is described in detail. As in Saccharomyces cerevisiae, gene replacement (also known as gene knock-out) methods provide a unique tool to investigate the function of specific P. pastoris genes. After construction, the function of the deleted gene is investigated from the phenotype of the mutant strain. In S. cerevisiae, an efficient polymerase chain reaction (PCR)-based method for the construction of gene replacement fragments has been developed. Modifications of this PCR method have been developed to adapt this approach to P. pastoris.

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References

  1. Cregg, J. M., Barringer, K. J., Hessler, A. Y., and Madden K. R. (1985) Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 5, 3376–3385.

    CAS  PubMed  Google Scholar 

  2. Cregg, J. M., Madden, K. R., Barringer, K. J., Thill, G. P., and Stillman, C. A. (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9, 1316–1323.

    CAS  PubMed  Google Scholar 

  3. Liu, H., Tan, X., Wilson, K., Veenhuis, M., and Cregg, J. M. (1995) PER3, a gene required for peroxisome biogenesis in Pichia pastoris, encodes a peroxisomal membrane protein involved in protein import. J. Biol. Chem. 270, 10,940–10,951.

    Article  CAS  PubMed  Google Scholar 

  4. Waterham, H. R., de Vries, Y., Russell, K. A., Xie, W., Veenhuis, M., and Cregg, J. M. (1996) The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to PAF-1. Mol. Cell. Biol. 16, 2527–2536.

    CAS  PubMed  Google Scholar 

  5. Johnson, M. A., Snyder, W. B., Lin-Cereghino, J., Veenhuis, M., Subramani, S., and Cregg, J. M. (2001) Pichia pastoris Pex14p, a phosphorylated peroxisomal membrane protein, is part of a PTS-receptor docking complex and interacts with many peroxins. Yeast 18, 621–641.

    Article  CAS  PubMed  Google Scholar 

  6. Snyder, W. B., Koller, A., Choy, A. J., et al. (1999) Pex17p is required for import of both peroxisome membrane and lumenal proteins and interacts with Pex19p and the peroxisomal targeting signal-receptor docking complex in Pichia pastoris. Mol. Biol. Cell 10, 4005–4019.

    CAS  PubMed  Google Scholar 

  7. Hinnen, A., Hicks, J. B., and Fink, G. R. (1978) Transformation of yeast. Proc. Natl. Acad. Sci. USA 75, 1929–1934.

    Article  CAS  PubMed  Google Scholar 

  8. Becker, D. M. and Guarante, L. (1991) High-efficiency transformation of yeast by electroporation. Methods Enzymol. 194, 182–187.

    Article  CAS  PubMed  Google Scholar 

  9. Lin-Cereghino, J., Wong, W. W., Xiong, S., Giang, W., Luong, L. T., Vu, J., Johnson, S. D., and Lin-Cereghino, G. P. (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. BioTechniques 38, 44–48.

    Article  CAS  PubMed  Google Scholar 

  10. Klebe, R. J., Harriss, J. V., Sharp, Z. D., and Douglas, M. G. (1983) A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25, 333–341.

    Article  CAS  PubMed  Google Scholar 

  11. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168.

    CAS  PubMed  Google Scholar 

  12. Pichia Expression Kit Instruction Manual, Version E, Invitrogen, San Diego CA, pp. 63.

    Google Scholar 

  13. Grey, M. and Brendel, M. (1995) Ten-minute electrotransformation of Saccharomyces cerevisiae, in Methods in Molecular Biology Vol. 47: Electroporation Protocols for Microorganisms, (Nickoloff, J. A. ed.), Humana Press, Totowa, NJ, pp. 269–272.

    Chapter  Google Scholar 

  14. Stowers, L., Gautsch, J., Dana, R., and Hoekstra, M. F. (1995) Yeast transformation and preparation of frozen spheroplasts for electroporation, in Methods in Molecular Biology Vol. 47: Electroporation Protocols for Microorganisms, (Nickoloff, J. A. ed.), Humana Press, Totowa, NJ, pp. 261–267.

    Chapter  Google Scholar 

  15. Lorow-Murray, D. and Jesse, J. (1991) High efficiency transformation of Saccharomyces cerevisiae by electroporation. Focus 13, 65–68.

    Google Scholar 

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© 2007 Humana Press Inc., Totowa, NJ

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Cregg, J.M. (2007). DNA-Mediated Transformation. In: Cregg, J.M. (eds) Pichia Protocols. Methods in Molecular Biology, vol 389. Humana Press. https://doi.org/10.1007/978-1-59745-456-8_3

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  • DOI: https://doi.org/10.1007/978-1-59745-456-8_3

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-429-6

  • Online ISBN: 978-1-59745-456-8

  • eBook Packages: Springer Protocols

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