A doxycycline-inducible gene deletion and marker recycling toolkit in Candida albicans

Abstract:

[Objective] To construct a fast, convenient and economical DNA manipulating toolkit for gene deletion and marker recycling in Candida albicans. [Methods] By using Exo Ⅲ-mediated ligase-independent cloning strategy, loxP sites were inserted into the flanking regions of heterologous selection markers CmLEU2, CdHIS1, and CdARG4, yielding the templates for marker cassettes amplification. The Tet-on promoter was assembled using a rTetR element which was codon-optimized and chemically synthesized. Codon-optimized recombinase Cre was placed downstream of the Tet-on promoter. Then this cassette was inserted into the CDS region of selection markers CdHIS1 and CdARG4, generating vectors for marker recycling. [Results] We constructed three loxP-flanked marker gene-containing vectors for gene deletion in C. albicans, and two vectors for marker recycling, containing the recombinase gene Cre under the control of the Tet-on promoter. [Conclusion] We successfully constructed an inducible gene deletion and marker recycling vector system in C. albicans, which was practically applied in gene deletion strain constructions. This system is helpful to construct single and multiple gene deletion strain constructions.

Key words:Candida albicans;gene deletion;Tet-on;marker recycling;Cre;loxP