Guisheng Zeng1, Yan-Ming Wang1, Fong Yee Chan1 & Yue Wang1,2
1 Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore.
2 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Published online in Nature Protocols on 30 January 2014.
The recent discovery of haploids in Candida albicans and the construction of tool strains carrying multiple auxotrophic markers have enabled, for the first time, performing one-step gene deletions in this fungal human pathogen. This breakthrough promises to greatly facilitate the molecular and genetic study of C. albicans biology and pathogenicity. However, the construction of gene-deletion mutants in C. albicans haploids involves many technical difficulties, particularly low transformation efficiency and autodiploidization. Here we describe a highly effective protocol for designing and performing one-step gene deletion in C. albicans haploids, which takes ~11 d to complete (not including plasmid construction, which may take ~2 weeks). A gene deletion cassette is constructed on a plasmid and subsequently released for transformation by lithium acetate incubation or electroporation. Desired gene-deletion mutants are identified and their ploidy is assessed simultaneously by colony PCR before final confirmation by flow cytometry.
Figure Legend: One-step targeted gene deletion in Candida albicans haploids. (a) Experimental workflow for one-step targeted gene deletion in C. albicans haploids. (b) Schematic diagram of the gene deletion construct. DNA sequences at the promoter and terminator regions of the target gene are PCR-amplified for cloning by using the primer pairs P01F/P02R (for fragment AB) and P03F/P04R (for fragment CD). Vector-derived primers T3 and T7 are used for sequencing to verify successful cloning. The construct is then digested with KpnI and SacII to release the gene deletion cassette for transformation. The primers used for colony PCR characterization of the transformants (URA3F, P05F and P06R) and the expected sizes of PCR products are indicated. (c) Ploidy analysis of transformants by flow cytometry. The PCR-characterized transformants (T1–T5) were cultured and cells were stained with propidium iodide for flow cytometry. Raw data were analyzed by the WinMDI (version 2.8) software. The DNA content of each transformant (purple) was compared with that of haploid (GZY803, blue) and diploid (SC5314, red) control strains.
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