| Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale
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Author:
Date:
2020-04-05
[Abstract] Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. ...
[摘要] [摘要 ] 人体肠道菌群中的玫瑰菌属和真细菌属在维持人类健康中起着重要作用，部分原因是产生丁酸盐，这是我们结肠上皮细胞的主要能源。但是，由于缺乏基因操作技术，我们对这些细菌的生物化学和生理学的认识受到限制。先前引入玫瑰花属物种的共轭转座子不容易被修饰，极大地限制了它们作为基因修饰平台的适用性。MOD ular质粒穿梭载体先前已经开发了用于梭菌物种，其与共享一个分类次序ř oseburia 和真杆菌，提高这些矢量可以在这些生物体中使用的可能性。在这里，我们描述了一种优化缀合协议使得能够自主复制的质粒的从转印大肠杆菌供体菌株为罗斯氏inulinivorans 和真杆菌rectale 。质粒的模块性质及其通过自主复制在受体细菌中得以维持的能力使其成为研究异源基因表达的理想之选，并成为其他遗传工具（包括反义RNA沉默或II 型移动子中断子基因破坏策略）的平台。
[背景 ] 玫瑰菌和真细菌属人类肠道菌群中含量最高的细菌（Zhernakova 等，2016），它们通过利用饮食和宿主衍生的多糖影响人类健康（Scott 等，2006和2011； Cockburn 等） 。，2015 ; 谢里登等人，2016 ）并产生促进健康的代谢物丁酸作为发酵终产物（邓肯等人，2002和2006） 。另外，这些物种能够通过鞭毛调节宿主免疫（Neville ...
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| In vitro DNA Polymerization Activity Assay Using Cell-free Extracts
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Author:
Date:
2014-08-20
[Abstract] This protocol has been designed to measure the in-vitro DNA polymerization activity in crude cell extracts of the Antarctic bacterium Pseudomonas syrinagae Lz4W. This bacterium can grow at 4 °C with optimum growth rate at 22 °C. The slow growth rate of the bacterium observed at low temperature (4 °C) compared to higher temperature (22 °C) can be attributed to the reduced rate of DNA replication at low temperature. Here we describe a protocol which we have used to quantify the in vitro DNA polymerization of cell extracts at two different temperatures.
[摘要] 该方案已经设计用于测量南极细菌假单胞菌syrinagae Lz4W的粗细胞提取物中的体外 DNA聚合活性。 该细菌可以在4℃下生长，在22℃以最佳生长速率生长。 在低温（4℃）下观察到的细菌与较高温度（22℃）相比的缓慢生长速率可以归因于在低温下DNA复制的速率降低。 在这里我们描述了一个协议，我们已经用来量化的体外细胞提取物在两个不同的温度下的DNA聚合。
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| 5-Hydroxymethylcytosine (5-hmC) Specific Enrichment
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Author:
Date:
2012-08-05
[Abstract] 5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA modification in mammalian genomes. This protocol is to be a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically, which could in turn be used for high-throughput mapping via next-generation sequencing.
[摘要] 5-羟甲基胞嘧啶（5-hmC）是哺乳动物基因组中新发现的DNA修饰。 这个协议是一种高效和选择性的化学方法来标记和捕获5-hmC，利用噬菌体酶，特别是添加葡萄糖部分到5-hmC，这可以反过来用于高通量映射通过下一个 - 生成测序。
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