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Parafilm

PARAFILM 膜

Company: Sigma-Aldrich
Catalog#: P7793
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Artificial Inoculation of Epichloë festucae into Lolium perenne, and Visualisation of Endophytic and Epiphyllous Fungal Growth
Author:
Date:
2018-09-05
[Abstract]  Natural hosts for the fungal endophyte Epichloë festucae include Festuca rubra (fine fescue) and Festuca trachyphylla (hard fescue). Some strains also form stable associations with Lolium perenne (perennial ryegrass). L. perenne is a suitable host to study fungal endophyte–grass interactions, such as endophytic fungal growth within the plant and epiphyllous growth on the plant surface. Here we provide a detailed protocol based on work by, for artificial inoculation of E. festucae into L. perenne, and newly developed staining and visualization techniques for observing endophytic and epiphyllous hyphae and the expressorium, an appressorium-like structure used by the fungus to exit the plant. The staining method uses a combination of ... [摘要]  真菌内生菌Epichloëfestucae的天然宿主包括 Festuca rubra (细羊茅)和 Festuca trachyphylla (硬羊茅)。一些菌株也与黑麦草(多年生黑麦草)形成稳定的关联。 升。 perenne 是研究真菌内生菌 - 草相互作用的合适宿主,例如植物内生真菌生长和植物表面的叶生生长。在这里,我们提供了一个基于工作的详细协议,用于 E的人工接种。 festucae 进入 L. perenne ,以及新开发的染色和可视化技术,用于观察内生和附生的菌丝和表达菌,这是一种类似真菌的结构,用于离开植物。染色方法使用葡聚糖结合苯胺蓝二铵盐(AB)和几丁质结合小麦胚凝集素缀合的Alexa Fluor ® 488-(WGA-AF488)的组合。该协议将是研究Epichloë -grass相互作用的有用工具,特别是不同Epichloë - 草关联,各种内生菌 - 宿主发育阶段的比较,以及分析突变Epichloë菌株。

【背景】Latch和Christensen(1985)开发了一种用Epichloë内生菌人工感染草的方案。这项工作一直是广泛的 E的基础。 festucae Fl1- L. perenne 共生研究(Scott et al。,2012年综述)。在我们最近的工作中,我们描述了一种新发现的真菌结构,即能够实现 E的表达式。 ...

Dual Fluorescence Reporter Based Analytical Flow Cytometry for miRNA Induced Regulation in Mammalian Cells
Author:
Date:
2018-09-05
[Abstract]  MicroRNA-induced gene regulation is a growing field in basic and translational research. Examining this regulation directly in cells is necessary to validate high-throughput data originated from RNA sequencing technologies. For this several studies employ luciferase-based reporters that usually measure the whole cell population, which comes with low resolution for the complexity of the miRNA-induced regulation. Here, we provide a protocol using a dual-fluorescence reporter and flow cytometry reaching single cell resolution; the protocol contains a simplified workflow that includes: vector generation, data acquisition, processing, and analysis using the R environment. Our protocol enables high-resolution measurements of miRNA induced post-transcriptional gene regulation and combined with ... [摘要]  MicroRNA诱导的基因调控是基础和转化研究中不断增长的领域。 直接在细胞中检查该调节对于验证源自RNA测序技术的高通量数据是必要的。 对于这一研究,一些研究采用基于荧光素酶的报告基因,通常测量全细胞群,其具有低分辨率的miRNA诱导调节的复杂性。 在这里,我们提供使用双荧光报告基因和流式细胞仪达到单细胞分辨率的方案; 该协议包含一个简化的工作流程,包括:使用R环境进行矢量生成,数据采集,处理和分析。 我们的协议可实现miRNA诱导的转录后基因调控的高分辨率测量,并结合系统生物学,可用于估计miRNA的熟练程度。

【背景】MicroRNAs(miRNA)是高度保守的小型非蛋白质编码RNA(21-22nt),可调节转录后基因表达并调节基因生物学过程,如发育和细胞稳态(Lagos-Quintana et al。,2001; Fabian et al。,2010; Bartel,2018),包括miRNA表达与肿瘤进展和侵袭性相关的几种病理(Lu et al。, 2005; Di Leva和Croce,2013; Krishnan et al。,2015; Bertoli et ...

Bacterial Competition Assay Based on Extracellular D-amino Acid Production
Author:
Date:
2018-04-05
[Abstract]  Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in fitness/survival of bacterial subpopulations within a community. Co-cultivation methods usually involve the growth of the competing bacteria in the same container. Therefore, within such mixed cultures the effect on growth caused by extracellular metabolites cannot be distinguished from direct physical interactions between species (e.g., T6SS effectors). However, this problem can be easily solved by using a filtration unit that allows free ... [摘要]  在激烈的竞争中,细菌生活在多种微生物群落中。为了坚持特定的生态位,许多物种会产生有毒的细胞外效应物作为干扰附近微生物生长的策略。这种效应子之一是非规范的D-氨基酸。在这里我们描述一种方法来测试D-氨基酸生产对社区内细菌亚群的适应/存活的影响。共培养方法通常涉及相同容器中竞争细菌的生长。因此,在这种混合培养物中,细胞外代谢物对生长的影响不能与物种间的直接物理相互作用区分开(例如T6SS效应物)。然而,通过使用允许小分解代谢物(例如L-和D-氨基酸)自由扩散的过滤单元可以容易地解决这个问题,同时将不同亚群保持在独立区室中。

通过这种方法,我们已经证明D-精氨酸是由霍乱弧菌产生的杀菌剂效应物,其强烈影响不同微生物亚群的存活。此外,D-精氨酸可作为混合菌群中的一种协同工具,用于保护非生产成员免受竞争细菌的侵害。

【背景】细菌生活在多种多样的物种共存并争夺现有资源的多种微生物群落中。细菌设计为在特定生态位持续存在的许多策略之一是产生有毒的细胞外代谢物作为干扰其他微生物生长和/或生存力的策略。已知D-氨基酸长时间在细菌培养物中具有细胞形状和活力的强大作用(Bopp,1965; Fox等人,1944; Kobayashi等人, 1948年; Yaw和Kakavas,1952年; Lark和Lark,1959年; Grula,1960年; ...

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