| A High-throughput Interbacterial Competition Platform
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Author:
Date:
2020-09-05
[Abstract] Contact-dependent interbacterial competition is a common strategy used by bacteria to fight for their ecological niches. Interbacterial competition is monitored by a competition assay involving co-culturing the attacker and the recipient bacterial cells on agar, followed by recovery of the surviving recipient cells. Conventional interbacterial competition assays rely on serial dilution, plate spreading, and colony counting experiments for the readout. The high demand for time and labor in a competition assay limits its use for large-scale screening. However, a high-throughput interbacterial competition screening method is required to screen genetic factors involved in an interbacterial competition. Here, using Agrobacterium tumefaciens as an attacker and Escherichia coli ...
[摘要] [摘要 ] 接触依赖性细菌间竞争是细菌争夺生态位的一种常用策略。通过竞争测定法监测细菌间竞争，该竞争测定法包括在琼脂上共同培养攻击者和受体细菌细胞，然后回收存活的受体细胞。常规的细菌竞争分析需要依靠连续稀释，平板铺展和菌落计数实验来进行读数。竞争测定中对时间和劳力的高需求限制了其在大规模筛选中的用途。然而，需要高通量的细菌间竞争筛选方法来筛选参与细菌间竞争的遗传因素。在这里，使用根癌土壤杆菌作为攻击者， 作为大肠埃希菌，我们开发了一种功能强大，快速，高效和高通量的VI型分泌系统依赖性细菌竞争筛选平台。该系统无需进行连续稀释和铺板即可进行96种同时竞争分析。该系统的数据分析仅依赖于直接和直接的菌落计数。该平台可以很容易地用于识别参与任何依赖接触的细菌竞争系统的新因素。
[背景 ] 细菌已经进化出多种策略来对抗其他细菌以获取生态位适应性，而接触依赖性细菌间竞争是广泛使用的策略之一（Granato 等人，2019）。细菌可以使用其I型（T1SS），IV型（T4SS），V型（T5SS），VI型（T6SS）或VII型（T7SS）分泌系统将蛋白毒素输送到其竞争细胞，受体细胞，并以接触依赖的方式杀死它（Aoki 等人，2005; Hood 等人，2010; Souza 等人，2015; Cao 等人，2016;García-Bayona ...
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| Mapping mRNA-18S rRNA Contacts Within Translation Initation Complex by Means of Reverse Transcriptase Termination Sites and RNAseq
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Author:
Date:
2020-08-20
[Abstract] The nucleotides involved in RNA-RNA interaction can be tagged by chemical- or UV-induced crosslinking, and further identified by classical or modern high throughput techniques. The contacts of mRNA with 18S rRNA that occur along the mRNA channel of 40S subunit have been mapped by site-specific UV crosslinking followed by reverse transcriptase termination sites (RTTS) using radioactive or fluorescent oligonucleotides. However, the sensitivity of this technique is restricted to the detection of those fragments that resulted from the most frequent crosslinkings. Here, we combined RTTS with RNAseq to map the mRNA-18S rRNA contacts with a much deeper resolution. Although aimed to detect the interaction of mRNA with the ES6S region of 18S rRNA, this technique can also be applied to map the ...
[摘要] [摘要 ] 可以通过化学或紫外线诱导的交联来标记参与RNA-RNA相互作用的核苷酸，并通过经典或现代的高通量技术对其进行进一步鉴定。沿着40S亚基的mRNA通道发生的18S rRNA与mRNA的接触已通过位点特异性UV交联进行了定位，随后使用放射性或荧光寡核苷酸进行了逆转录酶终止位点（RTTS）。但是，该技术的敏感性仅限于检测由最频繁的交联产生的那些片段。在这里，我们将RTTS与RNAseq结合使用，以更深的分辨率绘制了mRNA-18S rRNA接触图。尽管旨在检测mRNA与18S rRNA的ES6S区域的相互作用，但该技术也可以用于绘制mRNA与其他非编码RNA分子的相互作用（在转录，剪接或RNA介导的转录后调控过程中（例如，snRNA，microRNA和lncRNA）。
[背景 ] 是与非编码RNA的mRNA的相互作用volved mRNA中的生命周期的每个步骤，从它的生物合成和处理进入细胞核至细胞质中的翻译和最终降解。这些相互作用可以在大分子机器核糖体和剪接体，以及在较小的复合物如RISC或者发生（RNA诱导的沉默复合物）或lncRNA介导的基因表达调节期间（皮萨列夫等人，2008; Engreitz 。等人，2014 Sharma ...
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