Shuttle vectors for facile gap repair cloning and integration into a neutral locus in Candida albicans

Abstract

Candida albicans is the most prevalent fungal pathogen of humans. The current techniques used to construct C. albicans strains require integration of exogenous DNA at ectopic locations, which can exert position effects on gene expression that can confound the interpretation of data from critical experiments such as virulence assays. We have identified a large intergenic region, NEUT5L, which facilitates the integration and expression of ectopic genes. To construct and integrate inserts into this novel locus, we re-engineered yeast/bacterial shuttle vectors by incorporating 550 bp of homology to NEUT5L. These vectors allow rapid, facile cloning through in vivo recombination (gap repair) in Saccharomyces cerevisiae and efficient integration of the construct into the NEUT5L locus. Other useful features of these vectors include a choice of three selectable markers (URA3, the recyclable URA3-dpl200 or NAT1), and rare restriction enzyme recognition sites for releasing the insert from the vector prior to transformation into C. albicans, thereby reducing the insert size and preventing integration of non-C. albicans DNA. Importantly, unlike the commonly used RPS1/RP10 locus, integration at NEUT5L has no negative effect on growth rates and allows native-locus expression levels, making it an ideal genomic locus for the integration of exogenous DNA in C. albicans.

In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans

“…Correct integration at the NEUT5L locus was confirmed by PCR using primers NAT1-3118 and pDUP3-4969, as described by Gerami-Nejad et al (22). …”

The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia

“…1B). To construct HA-tagged torsinA and torsinA⌬E vectors, a single HA tag was introduced at the C terminus of torsinA and torsinA⌬E by in vivo recombination in S. cerevisiae following a previously published protocol (74). Briefly, primers LZJB12 and -13, encoding the HA tag sequence (Table 1), were annealed and co-transformed into S. cerevisiae together with NotI-digested pRS426GPD-torsinA or torsinA⌬E.…”

Zinc Cluster Transcription Factors Alter Virulence in Candida albicans

“…To complement ZCF15 deletion, we reintegrated ZCF15 using gap repair cloning in S. cerevisiae as described in Gerami-Nejad et al (2013). Briefly, ZCF15 ORF 6 650 bp was PCR amplified and cotransformed in S. cerevisiae BY4747 with BmgBI-digested pSN105 (Noble et al 2010).…”