Determination of Survival of Wildtype and Mutant Escherichia coli in Soil
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Author:
Date:
2017-07-20
[Abstract] E. coli resides in the gastrointestinal tract of humans and other warm-blooded animals but recent studies have shown that E. coli can persist and grow in various external environments including soil. The general stress response regulator, RpoS, helps E. coli overcome various stresses, however its role in soil survival was unknown. This soil survival assay protocol was developed and used to determine the role of the general stress response regulator, RpoS, in the survival of E. coli in soil. Using this soil survival assay, we demonstrated that RpoS was important for the survival of E. coli in soil. This protocol describes the development of the soil survival assay especially the recovery of E. coli inoculated into soil and can be ...
[摘要] 电子。 大肠杆菌位于人类和其他温血动物的胃肠道中,但最近的研究表明,E。 大肠杆菌可以在包括土壤在内的各种外部环境中持续生长。 一般的压力响应调节器RpoS有助于E。 克隆各种压力,但其在土壤中的作用尚不清楚。 开发了这种土壤生存测定方案,并用于确定一般应激反应调节剂RpoS在E的存活中的作用。 大肠杆菌在土壤中。 使用这种土壤生存测定,我们证明RpoS对于E的生存是重要的。 大肠杆菌在土壤中。 该方案描述了土壤生存测定的发展,特别是E的恢复。 大肠杆菌接种到土壤中,可适应于进一步研究土壤中其他细菌的存活。 【背景】大肠杆菌是属于肠杆菌科的革兰氏阴性兼性厌氧菌,其栖息于人类的肠道,温血动物和爬行动物(Berg,1996; Gordon和Cowling,2003)。可通过粪便通过水和沉淀物进行转运,并用作饮用水和娱乐用水中粪便污染的指标。使用 E。大肠杆菌作为粪便指标至少部分地基于其临时存在于主体胃肠道之外的假设(Ishii和Sadowsky,2008),并且在外部环境中长时间不存活。然而,一些研究已经分离出E。来自各种自然环境的大肠杆菌,例如城市废水,淡水,沙滩水,海滩沙土和土壤(Jiménez等人,1989; Brennan等人, 2010; Chiang等人,2011; Byappanahalli等人,2012; ...
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Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method
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Author:
Date:
2017-06-20
[Abstract] α-glucosidases (including maltases and isomaltases) are enzymes which release glucose from a set of α-glucosidic substrates. Their catalytic activity, substrate specificity and thermostability can be assayed using this trait. Thermostability of proteins can also be determined using a high-throughput differential scanning fluorometry method, also named Thermofluor. We have shown that Thermofluor can also be applied to predict binding of substrates and inhibitors to a yeast α-glucosidase. The methods described here in detail were used in Viigand et al., 2016.
[摘要] α-葡糖苷酶(包括麦芽糖酶和异麦芽糖酶)是从一组α-葡糖苷底物释放葡萄糖的酶。 可以使用该特征来测定其催化活性,底物特异性和热稳定性。 蛋白质的热稳定性也可以使用高通量差示扫描荧光测定法(也称为Thermofluor)来测定。 我们已经表明,Thermofluor也可以应用于预测底物和抑制剂与酵母α-葡萄糖苷酶的结合。 这里详细描述的方法用于Viigand等人,2016。 【背景】麦芽糖酶(EC 3.2.1.20)和异麦芽糖酶(EC 3.2.1.10)是根据CAZy分类属于糖苷水解酶家族13的α-葡糖苷酶(Lombard等,2014)。甲基营养酵母多形汉酵母的麦芽糖酶MAL1是非选择性的,它将产生D-葡萄糖的麦芽糖和异麦芽糖状α-葡萄糖苷水解为反应产物之一。因此,麦芽糖酶对其底物的活性可以根据葡萄糖释放来确定。该工作描述的葡萄糖液色辅助方法可以快速方便地测定麦芽糖酶的活性,底物特异性和热稳定性。重要的是,这种基于活性的方法可以适用于产生葡萄糖作为反应产物的其它酶。高通量Thermofluor方法主要用于蛋白质晶体学测量(热)稳定性蛋白质(Boivin等,2013; Ericsson等,2006)。我们使用Thermofluor ...
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