New pFA-cassettes for PCR-based gene manipulation in Candida albicans.

Several modules for efficient PCR-based gene disruption have recently been introduced in Candida albicans. These are based on auxotrophic marker genes for deficient strains derived from SC5314/CAI4. Commonly used protocols for the transformation C. albicans are based either on the lithium acetate procedure or on electroporation also used for Saccharomyces cerevisiae. Here we present our updated arsenal of pFA-modules that now include the heterologous marker genes HIS1 from C. dubliniensis and LEU2 from C. maltosa (Noble and Johnson 2005) and the dominant selection marker ca SAT1 (Reuss et al. 2004). We also introduce the Ashbya gossypii TEF1 -promoter as a strong constitutive promoter. With these new elements an enlarged collection of pFA-marker and pFA-marker-promoter modules were generated containing 17 new modules. In addition, N-terminal tagging with GFP-(GA) 6 and epitope-tagging modules using the 6 x-HIS-tag were constructed. This adds to the previous modules that only enabled C-terminal GFP-tagging of genes (Gola et al. 2003). In total 29 pFA-modules are currently freely available from our lab which - together with an update on the diagnostic verification procedure - further enlarge the C. albicans molecular toolbox and enhance our capabilities to use PCR-based gene alteration methods in C. albicans.