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Cassettes for PCR‐mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans
Author(s) -
GeramiNejad Maryam,
Berman Judith,
Gale Cheryl A.
Publication year - 2001
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.738
Subject(s) - yellow fluorescent protein , green fluorescent protein , biology , candida albicans , plasmid , gene , microbiology and biotechnology , aequorea victoria , homologous recombination , fluorescence , reporter gene , fluorescent protein , fluorescence microscope , yeast , genetics , gene expression , physics , quantum mechanics
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 . Copyright © 2001 John Wiley & Sons, Ltd.