Highly-efficient electrotransformation of the yeast Hansenula polymorpha View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

1994-04

AUTHORS

Klaas Nico Faber, Peter Haima, Wim Harder, Marten Veenhuis, Geert AB

ABSTRACT

A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7 x 10(6)/micrograms plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per microgram DNA) for H. polymorpha remained high when large amounts (up to 10 micrograms) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed. More... »

PAGES

305-310

Journal

TITLE

Current Genetics

ISSUE

4

VOLUME

25

Author Affiliations

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/bf00351482

DOI

http://dx.doi.org/10.1007/bf00351482

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1017142674

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/8082173


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38 schema:description A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7 x 10(6)/micrograms plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per microgram DNA) for H. polymorpha remained high when large amounts (up to 10 micrograms) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed.
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