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

Gerami-Nejad, Maryam, Zacchi, Lucia F., McClellan, Mark, Matter, Kathleen and Berman, Judith (2013) Shuttle vectors for facile cloning through gap repair and integration into a neutral locus in Candida albicans. Microbiology, 159 Part 3: 565-579. doi:10.1099/mic.0.064097-0


Author Gerami-Nejad, Maryam
Zacchi, Lucia F.
McClellan, Mark
Matter, Kathleen
Berman, Judith
Title Shuttle vectors for facile cloning through gap repair and integration into a neutral locus in Candida albicans
Formatted title
Shuttle vectors for facile cloning through gap repair and integration into a neutral locus in Candida albicans
Journal name Microbiology   Check publisher's open access policy
ISSN 1350-0872
1465-2080
Publication date 2013
Sub-type Article (original research)
DOI 10.1099/mic.0.064097-0
Open Access Status Not Open Access
Volume 159
Issue Part 3
Start page 565
End page 579
Total pages 15
Place of publication London, United Kingdom
Publisher The Microbiology Society
Language eng
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.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
 
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Created: Thu, 07 Apr 2016, 21:16:11 EST by Lucia Zacchi on behalf of School of Chemistry & Molecular Biosciences