Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.
Bibliographical noteFunding Information:
We thank N. Dean for providing the original mCherry FP sequence, M. Gerami-Nejad for construction of plasmids, B. Larson for technical assistance, and T. Heisel for helpful advice during the development of this project. J.B. was supported by the European Research Council Advanced Award 340087 (RAPLODAPT). Microscopy and imaging systems were provided by the University of Minnesota Pediatrics Foundation and the University of Minnesota Imaging Center.
© 2017 Journal of Visualized Experiments.
- Candida albicans
- Candida parapsilosis
- Fluorescent protein
- Issue 121
- Lithium acetate transformation
- PCR-mediated gene modification