Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants

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Microbial Pathogenesis (Impact Factor: 2). 03/2006; 40(2):69-81. DOI: 10.1016/j.micpath.2005.11.002
Source: PubMed

ABSTRACT The infectious yeast Candida albicans is a model organism for understanding the mechanisms of fungal pathogenicity. We describe the functional expression of the firefly luciferase gene, a reporter commonly used to tag genes in many other cellular systems. Due to a non-standard codon usage by this yeast, the CUG codons were first mutated to UUG to allow functional expression. When integrated into the chromosome of C. albicans with a strong constitutive promoter, cells bioluminesce when provided with luciferin substrate in their media. When fused to the inducible promoter from the HWP1 gene, expression and bioluminescence was only detected in cultures conditioning hyphal growth. We further used the luciferase gene as a selection to isolate transformed cell lines from clinical isolates of C. albicans, using a high-density screening strategy that purifies transformed colonies by virtue of light emission. This strategy requires no drug or auxotrophic selectable marker, and we were thus able to generate stable transformants of clinical isolates that are identical to the parental strain in all aspects tested, other than their bioluminescence. The firefly luciferase gene can, therefore, be used as a sensitive reporter to analyze gene function both in laboratory and clinical isolates of this medically important yeast.

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    • "luciferin distribution as a result of the severe clinical symptoms exhibited , as well as the presence of pulmonary lesions that may have been severe enough to restrict oxygen dispersion in the bronchoalveolar tree . In - deed , the researchers found a strong increase in light emis - sion when luciferin was directly administered to lungs ex vivo . Doyle et al . ( 2006b ) developed bioluminescent C . albicans using a codon optimized derivative of the firefly luciferase gene . The researchers found that while luciferase activity in protein extracts taken from C . albicans growing as yeast or hyphae were almost identical , light output from the hyphal stage was massively reduced ( Doyle et al . , 2006a )"
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