DOI: 10.5176/2251-2489_BioTech13.89
Authors: Wei-Chung Lai, Hsiao-Fang Sunny Sun, Jia-Ching Shieh
Abstract:
The bimolecular fluorescence complementation (BiFC) has been widely used in many model organisms for visualization of protein-protein interaction. To apply BiFC approach in Candida albicans, a Tet-on system with plasmids pWTN1 and pWTN2 was created first. Both plasmids carries Hygromycin B resistant marker (CaHygB) that is compatible with the original Tet-on system whose plasmid pNIM1 (Park & Morschhäuser, 2005) bearing nourseothricin resistant marker (CaSAT1). By using GFP as a reporter, the two complementary Tet-on systems appeared to function dosedependently and synergistically with doxycycline in C. albicans. To allow proteinprotein interaction detectable with visible spectrum, the yEmRFP (mCherry) red fluorescence protein was incorporated into our systems. DNA encoding each of the Nterminus (amino acid 1-159) and the C-terminus (amino acid 160-237) of yEmRFP was constructed into pWTN1 and pNIM1 to generate pWTN1-N and pNIM1-C, respectively. To verify the BiFC with yEmRFP in C. albicans, the known interactors of C. albicans Cdc42 (CaCdc42) and Rdi1 (CaRdi1) were used to create pWTN1-CDC42-N and pNIM1-C-RDI1. C. albicans cells carrying cassettes based on the two plasmids would co-express the N-terminal and C-terminal yEmRFP fused with CaCdc42 (CaCdc42-N) and CaRdi (C-CaRdi1), respectively, in the presence of doxycycline in which yEmRFP is functional due to interaction between CaCdc42 and CaRdi1. Colonies derived from these cells would be observed either as red fluorescence or light red under either fluorescence or visible spectrum. Establishment of the Tet-on based BiFC system in C. albicans should facilitate functional study and high-throughput screening of proteinprotein interaction in C. albicans.
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