title: Construction of Candida albicans Tet-on tagging vectors with a Ura-blaster cassette.
abstract: It has been difficult to develop molecular tools for studying the fungal pathogen Candida albicans because this species uses a non-standard genetic code and is diploid without a complete sexual cycle. Vector systems with regulatable promoters to produce conditional mutants, epitope tags for protein detection and recyclable selection markers are useful for functional study of genes. However, most currently available vectors contain only a subset of desired properties, which limits their application. To combine several useful properties in one vector, the vector pTET25 was initially modified into pTET25M, so that the URA3 gene flanked by dpl200 could be used repetitively. To enable more choices for cloning, a multiple cloning site was introduced at both ends of GFP in pTET25M. GFP expression was induced by doxycycline in a dose- and time-dependent manner when the plasmid was introduced into C. albicans with or without URA3. The applicability of the vectors was verified by constructing strains capable of expressing either the N-terminal GFP fusion of Cdc10 or the C-terminal GFP fusion of Cdc11. Additionally, by replacing the GFP gene of pTET25M with DNA sequence encoding Cdc10 with an epitope tag of six histidine residues at the C-terminus, doxycycline-induced expression of CDC10 was achieved when the expression vector was introduced into C. albicans. This new system allows for inducible expression of a desired C. albicans gene with the advantage of convenience of cloning. It also allows the presence of a recyclable URA3 marker and the detectable expression of fusion or epitope-tagged protein.
authors.affiliation: Department of Life Sciences, National Chung-Hsing University, Taichung City, Taiwan, Republic of China.
authors.forename: Wei-Chung
authors.initials: WC
authors.lastname: Lai
authors.forename: Tzu-Ling
authors.initials: TL
authors.lastname: Tseng
authors.forename: Ting
authors.initials: T
authors.lastname: Jian
authors.forename: Tai-Lin
authors.initials: TL
authors.lastname: Lee
authors.forename: Chun-Wen
authors.initials: CW
authors.lastname: Cheng
authors.forename: Jia-Ching
authors.initials: JC
authors.lastname: Shieh
mesh_terms: D002176:Candida albicans / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004318:Doxycycline / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D015533:Transcriptional Activation
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004318:Doxycycline