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Thiamine

盐酸硫胺素

Company: Sigma-Aldrich
Catalog#: T1270
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Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native Environment
Author:
Date:
2020-12-20
[Abstract]  

Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like platform to study these proteins in-situ using biophysical methods. Here, we describe in detail the preparation of OMVs from Escherichia coli, which are luminally enriched with periplasmic proteins and uniformly labeled with stable isotopes (2H and 15N), suitable for the subsequent characterisation of proteins at atomic resolution in their native environment by solution-state NMR spectroscopy. The ability to perform structural studies of periplasmic

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[摘要]  [摘要]细菌外膜囊泡(OMV)是由革兰氏阴性细菌的外膜出芽自然形成的。OMV由组成与原始外膜相同的脂质双层组成,并且在其内腔中含有周质成分。OMV富含特定的包膜蛋白,是使用生物物理方法原位研究这些蛋白的绝佳天然样平台。在这里,我们详细描述了从大肠杆菌制备OMV的方法,该方法在光亮时富含周质蛋白,并用稳定的同位素(2 H和15 N)均匀标记,适用于后续表征溶液状态NMR光谱分析天然环境中蛋白质的原子分辨率。执行周质成分的结构研究的能力,现场清除的方式来REAC兴的这种独特的细胞室的功能和机理细节的深入了解。

[背景]革兰氏阴性菌的周质是一个相当了不起的细胞室。这个空间中,内和外细菌膜之间禁闭,包含在抽蛋白一个ö ř dinarily高浓度超过300毫克毫升-1 (奥利弗,1996) ,并且在不存在的细胞来源,如ATP,功能几乎大力独立从其胞质对应物。到目前为止,有关周质蛋白的结构知识是使用从其天然环境分离的纯化蛋白专门获得的。因此,这种特殊环境可能对蛋白质施加的任何结构和功能影响在纯化过程中都会丢失。由于周质的体积比低,阻碍了使用生物物理方法如细胞内NMR光谱原位研究周质蛋白的努力,周质的体积比仅占细菌总体积的5-1 0%(Brass等,1986)。 )。

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Efficient Agrobacterium-mediated Transformation of the Elite–Indica Rice Variety Komboka
Author:
Date:
2020-09-05
[Abstract]  Genetic transformation is crucial for both investigating gene functions and for engineering of crops to introduce new traits. Rice (Oryza sativa L.) is an important model in plant research, since it is the staple food for more than half of the world’s population. As a result, numerous transformation methods have been developed for both indica and japonica rice. Since breeders continuously develop new rice varieties, transformation protocols have to be adapted for each new variety. Here we provide an optimized transformation protocol with detailed tips and tricks for a new African variety Komboka using immature embryos. In Komboka, we obtained an apparent transformation rate of up to 48% for GUS/GFP reporter gene constructs using this optimized protocol. This ... [摘要]  
[摘要 ] 遗传转化对于研究基因功能和农作物工程引入新性状均至关重要。水稻(Oryza sativa L.)是植物研究中的重要模型,因为它是世界一半以上人口的主食。其结果是,大量的转化方法已经开发了两个籼稻和粳稻米。由于育种者不断开发新的水稻品种,因此必须针对每个新品种适应转化方案。在这里,我们为使用未成熟胚的非洲新品种Komboka 提供了一种优化的转化协议,包括详细的技巧和窍门。在Komboka中,我们使用此优化方案对GUS / GFP报告基因构建体的表观转化率高达48%。该协议也适用于其他优质lite 稻品种。


[背景 ] 为植物的遗传转化的各种方法公顷一直在开发,例如穿孔(Shimamoto PEG介导的原生质体转染。等人,1989;达塔。等人,1992),生物射弹转化(Christou的等人,1991)和农杆菌-介导的转化(Slamet-Loedin 等,2014)。农杆菌介导的转化是将DNA引入植物的最广泛使用的方法之一(van Wordragen and Dons,1992)。该方法已被广泛用于研究,并已成为生物技术的关键先决条件。自从开发新的育种技术(如基因组编辑)以来,它就变得越来越重要(Char ...

MAMP-triggered Medium Alkalinization of Plant Cell Cultures
Author:
Date:
2020-04-20
[Abstract]  Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H+ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of ... [摘要]  [摘要 ] 植物识别多种微生物分子以检测和响应潜在的入侵者,通过细胞表面受体识别微生物相关的分子模式(MAMP)引发一系列生化反应,其中包括跨血浆的离子通量此类事件的后果是细胞外H + 离子浓度降低,这可以通过实验检测到植物细胞悬浮液中培养基pH值的变化,因此类似于活性氧(ROS)积累, MAP激酶的磷酸化和防御相关基因的诱导,MAMP诱导的培养基碱化可以用作激活植物免疫反应的代理。在这里,我们描述了测量烟草BY-2细胞培养基碱化的详细方案两种不同MAMP处理后的悬浮液:衍生自真菌细胞壁的壳六聚体(NAG6; N-乙酰氨基葡萄糖)和鞭毛蛋白表位flg22 ,在细菌鞭毛发现。氏S分析提供了可靠和快速的平台来访问毫安时触发免疫(在MTI)在烟草悬浮细胞,并且可以很容易地适应其他植物物种以及其他毫安。

[背景 ] 在整个进化过程中,植物都具有检测微生物衍生分子并建立起能够抓住有害相互作用的免疫反应的能力(Boutrot和Zipfel,2017年)。诱导此类免疫反应的微生物相关分子模式(MAMP)通常被广泛保存。微生物的结构成分,例如真菌的几丁质和细菌的鞭毛蛋白(Cook 等人,2015)。胞外植物受体对MAMP的识别会导致MAMP触发的免疫(MTI),从而阻止微生物入侵并维持植物健康。(Böhmet al。,2014)。

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