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Construction of Candida albicans Tet‐on tagging vectors with a Ura‐blaster cassette
Author(s) -
Lai WeiChung,
Tseng TzuLing,
Jian Ting,
Lee TaiLin,
Cheng ChunWen,
Shieh JiaChing
Publication year - 2011
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.1833
Subject(s) - ura3 , biology , multiple cloning site , candida albicans , plasmid , expression vector , gene , cloning vector , green fluorescent protein , fusion protein , cloning (programming) , computational biology , genetics , molecular cloning , microbiology and biotechnology , gene expression , recombinant dna , computer science , programming language
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 dpl 200 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. Copyright © 2010 John Wiley & Sons, Ltd.