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Phusion High-Fidelity DNA Polymerase (2 U/µL)

Phusion高保真DNA聚合酶(2 U /μL)

Company: Thermo Fisher Scientific
Catalog#: F530S
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Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
Author:
Date:
2017-08-20
[Abstract]  We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in silico-generated models used as search probes, were instrumental to succeeding in placing a large portion of the complex in the asymmetric unit. Electron density maps were then clear enough ... [摘要]  我们已经开发了产生组氨酸激酶DesK及其同源反应调节物DesR的位点特异性变体的方案,有助于捕获蛋白质的不同信号状态。两个合作伙伴在大肠杆菌中的共表达,确保调节剂过量,对于DesK:DesR复合物的可溶性生产和进一步纯化是至关重要的。通过使用分子置换的X射线晶体学解决了捕获在磷酸转移酶和磷酸酶反应步骤中的复合物的3D结构。该解决方案不是微不足道的,我们发现在用作搜索探针的硅片生成的模型中,有助于将大部分复合物放置在不对称单元中。电子密度图就足够清楚了,可以进行人工建模,获得完整的原子模型。这些方法有助于解决细菌信号领域的主要挑战,即获得稳定的激酶:调节复合物,具有不同的构象状态,适用于高分辨率晶体学研究。
【背景】关于细菌信号复合物,特别是双组分系统(TCS)的结构信息仍然很少(Casino et al。,2009; Gao and Stock,2009)。 TCS包含几乎所有细菌中的感觉组氨酸激酶(HK)和响应调节剂(RR)配偶体,它们允许细胞感知环境并通过适应性反应相应地反应。尽管在信号传输中这种切换机制的重要性(Trajtenberg等,2016),结构信息对于采用不同功能状态的TCS复合体甚至更为有限。我们研究了DesK-DesR途径(de Mendoza,2014),一种来自枯草芽孢杆菌的TCS,其参与调节细胞膜组成以适应降低双层流动性的线索,如冷休克。 ...

A Ribosome Footprinting Protocol for Plants
Author:
Date:
2016-11-05
[Abstract]  Ribosome footprinting, or Ribo-seq, has revolutionized the studies of translation. It was originally developed for yeast and mammalian cells in culture (Ingolia et al., 2009). Herein, we describe a plant-optimized hands-on ribosome footprinting protocol derived from previously published procedures of polysome isolation (Ingolia et al., 2009; Mustroph et al., 2009) and ribosome footprinting (Ingolia et al., 2009; Ingolia et al., 2013). With this protocol, we have been able to successfully isolate and analyze high-quality ribosomal footprints from different stages of in vitro grown Arabidopsis thaliana plants (dark-grown seedlings [Merchante et al., 2015] and 13-day-old plantlets in plates and plants grown in liquid ... [摘要]  核糖体足迹或Ribo-seq,彻底改变了翻译研究。它最初是为培养中的酵母和哺乳动物细胞开发的(Ingolia等人,2009)。本文中,我们描述了来自先前公开的多核糖体分离程序的植物优化的亲手核糖体印迹方案(Ingolia等人,2009; Mustroph等人,2009 )和核糖体印迹(Ingolia等人,2009; Ingolia等人,2013)。使用该协议,我们能够成功地分离和分析来自体外生长的拟南芥植物(黑暗生长的幼苗)的不同阶段的高质量核糖体印迹[Merchante < ,以及在液体培养物中生长的板和植物中的13天龄小植物[未发表的结果])。

[背景] 翻译在调节基因活性中的中心作用早已被公认,但是在响应于特定刺激的全基因组翻译定量变化的系统探索最近才变得技术上可行。最初为培养中的酵母和哺乳动物细胞开发的核糖体印迹技术(通常称为Ribo-seq)已经彻底改变了翻译调节和基因表达的研究,因为其允许确定核糖体在基因组 - ...

Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System
Author:
Date:
2014-09-05
[Abstract]  RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes CRISPR-Cas9 system, the Cas9 nuclease is directed by a short single guide RNA (gRNA or sgRNA) to generate double-strand breaks (DSB) at the specific sites of chromosomal DNA, thereby introducing mutations at the DSB by error-prone non-homologous end joining repairing. Cas9-gRNA recognizes targeted DNA based on complementarity between a gRNA spacer (~ 20 nt long leading sequence of gRNA) and its targeted DNA which precedes a protospacer-adjacent motif (PAM, Figure 1). In this ... [摘要]  使用细菌II型簇定期间隔的短回文重复序列(CRISPR)相关核酸酶(Cas)的RNA指导的基因组编辑(RGE)已经作为用于在包括植物和作物物种的许多生物体中的基因组编辑的简单和通用工具而出现。在基于化脓性链球菌CRISPR-Cas9系统的RGE中,Cas9核酸酶由短的单引导RNA(gRNA或sgRNA)引导以在染色体的特定位点产生双链断裂(DSB) DNA,从而通过易错的非同源末端连接修复在DSB处引入突变。 Cas9-gRNA基于gRNA间隔区(约20nt的gRNA的前导序列)与其在原间质体相邻基序(PAM,图1)之前的靶DNA之间的互补性识别靶向DNA。在该协议中,我们描述了使用CRISPR-Cas9系统和农杆菌介导的转化的植物RGE的一般程序。该协议包括gRNA设计,Cas9-gRNA质粒构建和水稻RGE的突变检测(基因分型),可适用于其他植物物种。

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