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Author:
Date:
2016-08-05
[Abstract] Anabaena sp. strain PCC 7120 has long served as a model organism for investigating N2-fixation, photosynthesis, and various plant-type metabolic pathways and biofuel production, as well as cellular differentiation (Xu et al., 2008, Halfmann et al., 2014, Golden and Yoon, 2003). Since more than 30,000 sequenced bacterial genomes are currently available (Land et al., 2015), specific gene inactivation and analyses of the corresponding mutant’s phenotype have become powerful tools in elucidating the function of a target gene. Here we describe a protocol to inactivate a target gene in Anabaena sp. PCC 7120 using a single-crossover approach. This approach requires only one-step cloning of an internal fragment of a target gene into an ...
[摘要] 菌株PCC 7120长期充当用于研究N 2 - 固定，光合作用和各种植物类型代谢途径和生物燃料生产以及细胞分化的模式生物体（Xu等人，/em>。，2008，Halfmann等人，2014，Golden and Yoon，2003）。由于目前可获得超过30,000个测序的细菌基因组（Land等人，2015），特异性基因失活和相应突变体表型的分析已成为阐明靶基因功能的有力工具。在这里，我们描述了灭活anabaena sp中的靶基因的方案。 PCC 7120使用单交叉方法。该方法仅需要将靶基因的内部片段一步克隆到整合载体中以产生货物质粒。在货物质粒和鱼腥藻染色体之间的单次交换（同源重组）时，内源靶基因通过产生3'-和5'-缺失的片段而被破坏。该基因失活方案基于整合载体pZR606（Chen等人，2015），其可以广泛应用于其他蓝细菌物种以及其他原核生物中的基因失活。
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Author:
Date:
2016-03-05
[Abstract] The displacement assay was designed to quantify the direct competition between two homologous ribosomal proteins from Mycobacterium tuberculosis, S18-1 and S18-2, for interaction with their cognate binding partner, ribosomal protein S6 (Prisic et al., 2015). The S18 proteins were dialyzed in two physiologically relevant conditions (i.e. in the presence of Zn2+ or with EDTA to chelate Zn2+) and then allowed to compete for binding to S6 which was maintained in limiting concentration. The result was obtained through an ELISA, where S6-His is first bound to a Ni2+-NTA plate, followed by addition of S18-2 in excess to S6, then by addition of increasing concentrations of S18-1. The percentage of S18-2 that remained bound to S6 was ...
[摘要] 设计置换测定以定量来自结核分枝杆菌（Mycobacterium tuberculosis）S18-1和S18-2的两个同源核糖体蛋白质之间的直接竞争，用于与它们的同源结合伴侣，核糖体蛋白S6（Prisic et al。，2015）。 S18蛋白质在两种生理相关条件下（即在Zn 2+ 2+存在下或用EDTA螯合Zn 2+ 2+）进行透析，并且然后允许竞争结合S6，其维持在有限浓度。通过ELISA获得结果，其中S6-His首先结合Ni 2+ 2+ -NTA板，然后加入超过S6的S18-2，然后加入递增浓度的S18-1。在化学发光ELISA中用S18-2蛋白和第二抗体特异性的抗体定量保留结合S6的S18-2的百分比。以这种方式，S18-1蛋白质被S18-1蛋白质的置换报告为通过用S18-2饱和S6实现的全强度信号的百分比。在其基础上，该方法利用天然蛋白质 - 蛋白质相互作用，并且可以应用于其中两种或更多种蛋白质竞争结合上述靶配体的其他系统。
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