Healey Kelley R, Singh Ashutosh
Department of Biology, William Paterson University, Wayne, NJ, USA.
Medical Mycology Unit, Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India.
Methods Mol Biol. 2023;2658:181-190. doi: 10.1007/978-1-0716-3155-3_12.
Understanding how a gene variant may influence antifungal resistance, or other phenotypic characteristics, is an important step in determining or dissecting resistance mechanisms. The influence of specific genes or gene alleles on a phenotype can initially be assessed within the model organism, Saccharomyces cerevisiae. S. cerevisiae exhibits efficient rates of homologous recombination making it amendable for heterologous expression and represents a susceptible organism that can be used to determine changes in antifungal susceptibilities. Many groups have developed different methodologies for the cloning, expression, and screening processes. In this chapter, we present straightforward methodology that utilizes gap-repair cloning to express a plasmid-borne copy of Candida auris ERG11 within S. cerevisiae. Multiple alleles can be compared in order to determine how specific alterations influence triazole susceptibility. Primer design, gap-repair co-transformation, and colony PCR screening are detailed.
了解基因变异如何影响抗真菌耐药性或其他表型特征，是确定或剖析耐药机制的重要一步。特定基因或基因等位基因对表型的影响最初可在模式生物酿酒酵母中进行评估。酿酒酵母表现出高效的同源重组率，使其适合异源表达，并且代表了一种可用于确定抗真菌药敏性变化的敏感生物。许多研究小组已经开发出不同的克隆、表达和筛选方法。在本章中，我们介绍了一种直接的方法，该方法利用缺口修复克隆在酿酒酵母中表达耳念珠菌ERG11的质粒携带拷贝。可以比较多个等位基因，以确定特定改变如何影响三唑敏感性。详细介绍了引物设计、缺口修复共转化和菌落PCR筛选。
