Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans

Maryam Gerami-Nejad, Judith Berman, Cheryl A. Gale

Research output: Contribution to journalArticlepeer-review

166 Scopus citations

Abstract

We have developed a set of plasmids containing fluorescent protein cassettes for use in PCR-mediated gene tagging in Candida albicans. We engineered YFP and CFP variants of the GFP sequence optimized for C. albicans codon usage. The fluorescent protein sequences, linked to C. albicans auxotrophic marker sequences, were amplified by PCR and transformed directly into yeast. Gene-specific sequence was incorporated into the PCR primers, such that the tag-cassette integrates by homologous recombination at the 3′-end of the gene of interest. This technique was used to tag Cdc3 and Tub1 with GFP, YFP and CFP, which were readily visualized by fluorescence microscopy and localized as expected. In addition, Tub1-YFP and Cdc3-CFP were visualized in the same cells. Thus, this technique directs one-step construction of multiple fluorescent protein fusions, facilitating the study of protein co-expression and co-localization in C. albicans cells in vivo.

Original languageEnglish (US)
Pages (from-to)859-864
Number of pages6
JournalYeast
Volume18
Issue number9
DOIs
StatePublished - 2001

Keywords

  • Candida albicans
  • Cyan fluorescent protein
  • Epitope tagging
  • Green fluorescent protein
  • Polymerase chain reaction
  • Protein co-localization
  • Yellow fluorescent protein

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