Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants
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 )"
ABSTRACT: According to World Health Organization estimates, infectious organisms are responsible for approximately one in four deaths worldwide. Animal models play an essential role in the development of vaccines and therapeutic agents but large numbers of animals are required to obtain quantitative microbiological data by tissue sampling. Biophotonic imaging (BPI) is a highly sensitive, nontoxic technique based on the detection of visible light, produced by luciferase-catalysed reactions (bioluminescence) or by excitation of fluorescent molecules, using sensitive photon detectors. The development of bioluminescent/fluorescent microorganisms therefore allows the real-time noninvasive detection of microorganisms within intact living animals. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, reducing the number of animals required to yield statistically meaningful data. In the study of infectious disease, the use of BPI is becoming widespread due to the novel insights it can provide into established models, as well as the impact of the technique on two of the guiding principles of using animals in research, namely reduction and refinement. Here, we review the technology of BPI, from the instrumentation through to the generation of a photonic signal, and illustrate how the technique is shedding light on infection dynamics in vivo.FEMS microbiology reviews 09/2010; 35(2):360-94. DOI:10.1111/j.1574-6976.2010.00252.x · 13.81 Impact Factor
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ABSTRACT: Novel luciferase reporters have been developed that allow real-time monitoring of infections by the fungal pathogens Candida albicans and Aspergillus fumigatus. Although these reporters still suffer limitations in the context of invasive infections, they provide unprecedented tools to monitor superficial infections and the efficacy of antifungal drugs or vaccines. In particular, the sensitivity and ease of detection of the cell-surface Gaussia princeps luciferase developed for C. albicans should make it a powerful tool for functional genomics studies in this and other pathogenic fungi.Virulence 10/2014; 1(3):174-6. DOI:10.4161/viru.1.3.11119 · 3.32 Impact Factor
Conference Paper: Effects of environment and aging upon missile reliability[Show abstract] [Hide abstract]
ABSTRACT: Storage test data collected over 15 years of the United States Navy Harpoon anti-ship missile program provides experiential data to compare to theoretical models. The Harpoon missile shows an increasing storage MTBF with an increasing missile population age. Electronic subassemblies show storage MTBF growth with time. Mechanical subassemblies have higher storage MTBFs, but show little or no growth. Air-launched, ship-launched, and submarine-launched variants of the Harpoon missile are exposed to different environments which causes variations in the resulting storage reliabilities. The effects upon storage reliability apparently are not related to temperature and humidity so much as they are related to vibration and physical shockReliability and Maintainability Symposium, 1998. Proceedings., Annual; 02/1998