| Evaluation of Plasmid Stability by Negative Selection in Gram-negative Bacteria
|
|
Author:
Date:
2017-05-05
[Abstract] Plasmid stability can be measured using antibiotic-resistance plasmid derivatives by positive selection. However, highly stable plasmids are below the sensitivity range of these assays. To solve this problem we describe a novel, highly sensitive method to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells. The assay proposed here is based on an aph-parE cassette. When synthesized in the cell, the ParE toxin induces cell death. ParE synthesis is controlled by a rhamnose-inducible promoter. When bacteria carrying the aph-parE module are grown in media containing rhamnose as the only carbon source, ParE is synthesized and plasmid-containing cells are eliminated. Kanamycin resistance (aph) is ...
[摘要] 可以通过阳性选择使用抗生素抗性质粒衍生物来测量质粒稳定性。然而，高度稳定的质粒低于这些测定的灵敏度范围。为了解决这个问题，我们描述了一种新颖的，高度灵敏的方法来测量质粒稳定性，这是基于在含有质粒的细胞消除后，无质粒细胞的选择。这里提出的检测方法是基于aph-parE 盒式磁带。当细胞合成时，ParE毒素诱导细胞死亡。 ParE合成由鼠李糖诱导型启动子控制。当携带 aph-parE 模块的细菌生长在含有鼠李糖作为唯一碳源的培养基中时，合成ParE，消除含有质粒的细胞。进一步使用卡那霉素抗性（ aph ）来证实在鼠李糖生长的细菌中不存在质粒。
背景 通过使用抗生素抗性质粒衍生物的阳性选择测定质粒稳定性。携带研究质粒的细胞在选择性抗生素（Gerdes等人，1985; del Solar等人，1987）的存在下被阳性选择。这种技术的主要缺点是其灵敏度;高度稳定的质粒低于这些测定的敏感性。为了解决这个问题，已经描述了依靠直接选择无质粒细胞例如 - 四环素系统的替代方法（Bochner等人，1980; Maloy和Nunn，1981; Garcia-Quintanilla 等人，2006）。 - ...
|
|
|
| Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds
|
|
Author:
Date:
2016-05-05
[Abstract] The Arabidopsis thaliana seed coat epidermis produces copious amounts of mucilage polysaccharides (Haughn and Western, 2012). Characterization of mucilage mutants has identified novel genes required for cell wall biosynthesis and modification (North et al., 2014). The biochemical analysis of seed mucilage is essential to evaluate how different mutations affect cell wall structure (Voiniciuc et al., 2015c). Here we describe a robust method to screen the monosaccharide composition of Arabidopsis seed mucilage using ion chromatography (IC). Mucilage from up to 48 samples can be extracted and prepared for IC analysis within 24 h (only 4 h hands-on). Furthermore, this protocol enables fast separation (31 min per sample), automatic detection and ...
[摘要] 拟南芥种皮表皮产生大量的粘液多糖（Haughn和Western，2012）。 粘液突变体的表征已经鉴定了细胞壁生物合成和修饰所需的新基因（North等人，2014）。 种子粘液的生物化学分析对于评估不同突变如何影响细胞壁结构是必要的（Voiniciuc等人，2015c）。 在这里我们描述了使用离子色谱（IC）筛选拟南芥种子粘液的单糖组成的有力方法。 在24小时内（仅4小时手动）可以提取并准备来自多达48个样品的粘蛋白并进行IC分析。 此外，该协议使快速分离（每个样品31分钟），自动检测和定量的中性和酸性糖。
|
|
|
| Quantification of the Mucilage Detachment from Arabidopsis Seeds
|
|
Author:
Date:
2016-05-05
[Abstract] The Arabidopsis thaliana seed coat produces large amounts of cell wall polysaccharides, which swell out of the epidermal cells upon hydration of the mature dry seeds. While most mucilage polymers immediately diffuse in the surrounding solution, the remaining fraction tightly adheres to the seed, forming a dense gel-like capsule (Macquet et al., 2007). Recent evidence suggests that the adherence of mucilage is mediated by complex interactions between several cell wall components (Griffiths et al., 2014; Voiniciuc et al., 2015a). Therefore, it is important to evaluate how different cell wall mutants impact this mucilage property. This protocol facilitates the analysis of monosaccharides in sequentially extracted mucilage fractions, and quantifies the ...
[摘要] 拟南芥种子产生大量的细胞壁多糖，其在成熟干种子水合时从表皮细胞中膨胀。 虽然大多数粘液聚合物立即在周围溶液中扩散，但剩余部分紧密粘附于种子，形成致密的凝胶状胶囊（Macquet等人，2007）。 最近的证据表明粘液的粘附由几个细胞壁组分之间的复杂相互作用介导（Griffiths等人，2014; Voiniciuc等人，2015a）。 因此，重要的是评估不同的细胞壁突变体如何影响这种粘液性质。 该协议有助于分析顺序提取的粘液部分中的单糖，并且量化每种成分从种子中的脱离。
|
|
|