| Super-resolution Imaging of the T cell Central Supramolecular Signaling Cluster Using Stimulated Emission Depletion Microscopy
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Author:
Date:
2020-11-05
[Abstract] Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4+ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist ...
[摘要] [摘要]超分子信号组装体因其独特的信号传导特性而受到关注。在抗原呈递细胞（APC）激活的T细胞的中心界面处形成一个微米级的信号传导组件，即中央超分子信号簇（cSMAC ）。用于激活T细胞（LAT）的衔接子蛋白接头是关键的cSMAC组件。所述CSMAC已被广泛使用的CD4全内反射荧光显微镜研究+由平面APC替代活化的T细胞。在这里，我们提供了一种协议，可以在T细胞和APC之间的接口在其细胞上下文中成像cSMAC 。超分辨率激发发射耗尽显微镜（STED）用于确定LA T的定位，其活性，磷酸化形式及其整个池的位置。在固定和抗体染色之前，将载有激动剂肽的APC与TCR转基因CD4 + T细胞孵育4.5分钟。固定的细胞对在Leica SP8 AOBS共聚焦激光扫描显微镜上使用100x 1.4 NA物镜成像。LAT聚集在多个超分子复合物中，并确定了它们的数量和大小分布。使用此协议，可以量化在T细胞和APC之间的界面在其细胞环境中的cSMAC属性。
[背景] ...
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| In vivo Quantification of Alkanes in Escherichia coli
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Author:
Date:
2020-04-20
[Abstract] Microbial production of alkanes employing synthetic biology tools has gained tremendous attention owing to the high energy density and similarity of alkanes to existing petroleum fuels. One of the most commonly studied pathways includes the production of alkanes by AAR (acyl-ACP (acyl carrier protein) reductase)-ADO (aldehyde deformylating oxygenase) pathway. Here, the intermediates of fatty acid synthesis pathway are used as substrate by the AAR enzyme to make fatty aldehyde, which is then deformylated by ADO to make linear chain alkane. However, the variation in substrate availability to the first enzyme of the pathway, i.e., AAR, via fatty acid synthesis pathway and low turnover of the ADO enzyme make calculation of yields and titers under in vivo conditions extremely ...
[摘要] [摘要] 由于烷烃的高能量密度和与现有石油燃料的相似性，使用合成生物学工具生产烷烃的微生物受到了广泛关注。最常研究的途径之一是通过AAR（酰基-ACP （酰基）载体蛋白）还原酶）-ADO（醛Deformylating 加氧酶）途径。在这里，中间体脂肪酸合成途径被用作基材由AAR Enzym E要使脂肪醛，然后是Deformylated 通过ADO，使线性链烷烃。但是，即该途径的第一种酶的底物利用率的变化，即，AAR，通过脂肪酸合成途径和ADO酶的低周转率，使得在体内条件下的产量和效价的计算极为困难。在体内测定中，将确定的ADO酶底物外加到培养基中有助于监测菌体的流入。因此，该底物提供了更准确的产物收率测量方法。在此方案中，我们包括用于实施体内测定法以监测大肠杆菌中烷烃生产的详细指南。
[背景] 利用工程微生物生产烷烃的研究已广受欢迎，因为它提供了一种有吸引力的替代方案，可减少对化石燃料的依赖，同时减轻气候变化的影响（Lee 等，2008；Knothe ，2010； Lu，2010； Schirmer 等。人，2010;谭等人。，2011）各种途径已被发现或人工Assemb ...
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