Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans.
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.
Gerami-Nejad
M
M
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Berman
J
J
Gale
C A
CA
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D002176:Candida albicans / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D015203:Reproducibility of Results; D013698:Templates, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins