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Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus

机译:遗传工具,以增强金黄色葡萄球菌的基因功能和调控的研究

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The bursa aurealis transposon has been used to create transposon insertion libraries of Bacillus anthracis and Staphylococcus aureus . To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessential S. aureus genes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study of S. aureus .
机译:金黄色葡萄囊转座子已被用于创建炭疽芽孢杆菌和金黄色葡萄球菌的转座子插入库。为了提供一套遗传工具来增强这些文库的效用,我们生成了一个等位基因交换系统,该系统允许用有用的遗传标记和荧光报告基因替换转座子。这些工具在内布拉斯加转座子突变体库(NTML)中进行了测试,其中包含在1952个非必需金黄色葡萄球菌基因中定义的转座子插入。首先,我们生成了一个质粒,使研究人员可以用非编码DNA片段替换转座子中编码绿色荧光蛋白(GFP)和红霉素抗性的基因,从而在染色体内留下无标记的突变。其次,我们生产了等位基因交换质粒,用编码四环素,卡那霉素和壮观霉素抗性的替代抗生素抗性盒替代转座子,从而可以同时选择多个染色体突变。第三,我们生成了一系列荧光报告基因构建体,在等位基因交换后,它们会生成编码密码子优化的增强型青色荧光蛋白(ECFP),增强型黄色荧光蛋白(EYFP),DsRed.T3(DNT)和eqFP650的转录报告子。以及超级文件夹绿色荧光蛋白(sGFP)。总体而言,将NTML与该等位基因交换系统结合在一起,为金黄色葡萄球菌的研究提供了无与伦比的资源。

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