Name: ms2-affinity purification coupled with rna sequencing in gram-positive bacteria
Uploaded: 2021-02-23T15:00:00
Description: Watch this Scientific Journal Video about MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria at JoVE.com

这项工作得到了"国家重建联盟"（ANR，授予ANR-16-CE11-0007-01，RIBOSTAPH，和ANR-18-CE12-0025-04，科诺科，公关）的支持。它还在实验室交易所 NetRNA ANR-10-LABX-0036 和 ANR-17-EURE-0023（对 PR）的框架下发布，作为未来项目投资的一部分，由 ANR 管理的国家提供资金。DL得到了欧盟Horizna 2020研究和创新计划的支持，该计划是根据玛丽·斯考多斯卡-库里赠款协议第753137-萨纳雷格号。E. Massé实验室的工作得到了加拿大卫生研究所（CIHR）、加拿大自然科学和工程研究理事会（NSERC）和国家卫生研究院NIH团队赠款R01 GM092830-06A1的运营赠款的支持。

1. 缓冲区和媒体
<ol><li>对于 MAPS 实验，请准备以下缓冲区和媒体： - 缓冲区A（150毫克，20米三轮车-HCl pH 8，1毫米M毫克2 和1米DTT） - 缓冲E（250毫克，20米三轮车-HCl pH 8，12米麦芽糖，0.1%特里顿，1毫克2 和1毫克DTT） - RNA加载缓冲区（0.025%二甲苯氰醇和0.025%溴酚蓝色在8M尿素） - 脑心输液 （BHI） 介质 （12.5 g 小牛脑， 10 克肽， 5 克牛肉心脏， 5 克 NaCl， 2.5 克纳...

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革兰氏阳性细菌的修改协议 MAPS的初始协议是为了研究大肠杆菌20、30模型中的sRNA相互作用而开发的。在这里，我们描述了一个修改后的协议，它适用于机会性人类病原体金黄色葡萄球菌中依赖sRNA的监管网络的特征，并且肯定可转化为其他Gram阳性细菌，病原学与否。
特别注意细胞裂解步骤。法国的印刷?...

在大多数细菌基因组中，已经发现了数百种，甚至数千种小型调节RNA（SRNA），但其中绝大多数的功能仍然不寻常。总的来说，sRNA是短的非编码分子，在细菌生理学和适应波动环境1，2，3中起着主要作用。事实上，这些大分子是众多复杂监管网络的中心，影响代谢途径、应激反应，但也影响毒性和抗生素耐药性。从逻辑上讲，它们的合成是由特定的环境刺激（例如营养饥饿、氧化或膜应力）触发的。大多数 sRNA 通过短和非连续基配对在转录后级别调节多个目标 mRNA。他们通常通过与核糖体竞争翻译启动区域4来阻止翻译的启动。sRNA：mRNA复式的形成也常常导致目标mRNA通过招募特定的RNA而主动退化。
sRNA 靶点（即其目标 RNA 的整组）的定性允许识别其干预的代谢通路及其回答的潜在信号。因此，特定 sRNA 的功能通常可以从其目标中推断出。为此，已经开发了几个西里科预测工具，如因塔纳和科普拉纳5，6，7。它们特别依赖于序列互补性、配对能量和潜在交互站点的可访问性，以确定假定 sRNA 合作伙伴。但是，预测算法并不集成影响体内基础配对的所有因素，例如 RNA 陪护8的参与，这些因素有利于次优交互或双方的共同表达。由于其固有的局限性，预测工具的误报率仍然很高。大多数实验性的大规模方法是基于sRNA的共纯化：mRNA夫妇与标记RNA结合蛋白（RBP）6，9相互作用。例如，通过利格化和测序（RIL-seq）方法识别出RNA复式与RNA伴郎（如大肠杆菌10、11中的Hfq和ProQ）共同纯化。一种类似的技术称为紫外线交叉链接，利化和混合测序（CLASH）被应用到大肠杆菌12，13的RNA和Hfq相关sRNA。尽管Hfq和ProQ在多种细菌8、14、15的sRNA介介调节中的作用十分突出，但在像S.Aureus16、17、18这样的几个生物体中，基于sRNA的调节似乎与RNA无关。即使像沃特斯和同事13所证明的那样，与RNA关联的RNA复式净化是可行的，但这种情况仍然很棘手，因为RNA会引发它们的快速退化。因此，MS2-亲和力纯化加上RNA测序（MAPS）方法19，20构成了这种生物体的坚实替代品。
与上述方法不同，MAPS 使用特定的 sRNA 作为诱饵来捕获所有相互作用的RNA，因此不依赖于 RBP 的参与。整个过程在 图1中描述。简言之，sRNA 标记为 5' 与 MS2 RNA 贴机，该贴子由 MS2 涂层蛋白特别识别。这种蛋白质与麦芽糖结合蛋白（MBP）融合，固定在淀粉酶树脂上。因此，MS2-sRNA 及其 RNA 合作伙伴保留在亲和力色谱列中。与麦芽糖分离后，使用高通量RNA测序进行联合纯化RNA识别，然后进行生物信息分析（图2）。MAPS 技术最终绘制了一张内部发生的所有潜在交互的交互映射图。
MAPS技术最初是在非致病性革兰阴性大肠杆菌21中实施的。值得注意的是，MAPS 帮助识别了一个 tRNA 衍生片段，该片段与 RyhB 和 Rybb sRNA 特别交互，并防止任何 sRNA 转录噪声在非诱导条件下调节 mRNA 目标。此后，MAPS 已成功应用于其他大肠杆菌sRNA，如 DsrA22、RprA23、CyaR23和 GcvB24（表 1）。除了确认以前已知的目标外，MAPS 还扩展了这些众所周知的 sRNA 的目标范围。最近，MAPS已经在沙门氏菌蒂菲穆里姆进行，并透露，SraL sRNA绑定到rho mRNA，编码转录终止因子25。通过这种配对，SraL 保护rho mRNA 免受由 Rho 本身触发的过早转录终止。有趣的是，该技术不限于 sRNA，可以应用于任何类型的蜂窝RNA，例如使用 tRNA 衍生的片段26和 mRNA22的 5'未翻译区域 （表 1）。
MAPS方法也已适应致病性革兰阳性细菌S.金黄色葡萄球菌19。具体来说，裂解协议已被广泛修改，以有效地打破细胞，由于比Gram阴性细菌更厚的细胞壁，并保持RNA完整性。这个经过调整的协议已经解开了RSA27、RsaI28和RSAC29的相互作用。这种方法深入了解了这些 sRNA 在细胞表面特性、葡萄糖吸收和氧化应激反应的调控机制中的关键作用。
2015年在 大肠杆菌 中制定和实施的协议最近被详细描述了30。在这里，我们提供修改后的 MAPS 协议，该协议特别适合研究革兰阳性（较厚的细胞壁）细菌中的 sRNA 监管网络，无论是非致病细菌还是致病菌（安全预防措施）。

<table border="0" cellpadding="0" cellspacing="0" style="width:834px;" width="834">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">1.5 mL microcentrifuge tube</td>
			<td style="width:187px;">Sarstedt</td>
			<td style="width:119px;">72.690.001</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">15 mL centrifuge tubes</td>
			<td style="width:187px;">Falcon</td>
			<td style="width:119px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">2 mL microcentrifuge tube</td>
			<td style="width:187px;">Starstedt</td>
			<td style="width:119px;">72.691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">2100 Bioanalyzer Instrument</td>
			<td style="width:187px;">Agilent</td>
			<td style="width:119px;">G2939BA</td>
			<td>RNA quantity and quality</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">250 mL culture flask</td>
			<td style="width:187px;">Dominique Dutscher</td>
			<td style="width:119px;">2515074</td>
			<td>Bacterial cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">50 mL centrifuge tubes</td>
			<td style="width:187px;">Falcon</td>
			<td style="width:119px;">352051</td>
			<td>Culture centrifugation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absolute ethanol</td>
			<td style="width:187px;">VWR Chemicals</td>
			<td style="width:119px;">20821.321</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Allegra X-12R Centrifuge</td>
			<td style="width:187px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td>Bacterial pelleting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Ampicilin (amp)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9518-5G</td>
			<td>Growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Amylose resin</td>
			<td style="width:187px;">New England BioLabs</td>
			<td style="width:119px;">E8021S</td>
			<td>MS2-affinity purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Anti-dioxigenin AP Fab fragment</td>
			<td style="width:187px;">Sigma Aldrich</td>
			<td style="width:119px;">11093274910</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoradiography cassette</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">50-212-726</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">BamHI</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">ER0051</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">BHI (Brain Heart Infusion) Broth</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">53286</td>
			<td>Growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Blocking reagent</td>
			<td style="width:187px;">Sigma Aldrich</td>
			<td style="width:119px;">11096176001</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">CDP-Star</td>
			<td style="width:187px;">Sigma Aldrich</td>
			<td style="width:119px;">11759051001</td>
			<td>Northern blot assays (substrate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Centrifuge 5415 R</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Chloroform</td>
			<td style="width:187px;">Dominique Dutscher</td>
			<td style="width:119px;">508320-CER</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">DIG-RNA labelling mix</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">11277073910</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">DNase I</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">4716728001</td>
			<td>DNase treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erythromycin (ery)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td>Fluka 45673</td>
			<td>Growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPrep device</td>
			<td style="width:187px;">MP Biomedicals</td>
			<td style="width:119px;">116004500</td>
			<td>Mechanical lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidium Thiocyanate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9277-250G</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Hybridization Hoven Hybrigene</td>
			<td style="width:187px;">Techne</td>
			<td style="width:119px;">FHB4DD</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Hybridization tubes</td>
			<td style="width:187px;">Techne</td>
			<td style="width:119px;">FHB16</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoamyl alcohol</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">A/6960/08</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">LB (Lysogeny Broth)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">L3022</td>
			<td>Growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysing Matrix B Bulk</td>
			<td style="width:187px;">MP Biomedicals</td>
			<td style="width:119px;">6540-428</td>
			<td>Mechanical lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">MicroPulser Electroporator</td>
			<td style="width:187px;">BioRad</td>
			<td style="width:119px;">1652100</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Milli-Q water device</td>
			<td style="width:187px;">Millipore</td>
			<td style="width:119px;">Z00QSV0WW</td>
			<td>Ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">NanoDrop spectrophotometer</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;"></td>
			<td>RNA/DNA quantity and quality</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Nitrocellulose membrane</td>
			<td style="width:187px;">Dominique Dutsher</td>
			<td style="width:119px;">10600002</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Phembact Neutre</td>
			<td style="width:187px;">PHEM Technologies</td>
			<td style="width:119px;">BAC03-5-11205</td>
			<td>Cleaning and decontamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Phenol</td>
			<td style="width:187px;">Carl Roth</td>
			<td style="width:119px;">38.2</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Phusion High-Fidelity DNA Polymerase</td>
			<td style="width:187px;">New England Biolabs</td>
			<td style="width:119px;">M0530</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">pMBP-MS2</td>
			<td style="width:187px;">Addgene</td>
			<td style="width:119px;">65104</td>
			<td>MS2-MBP production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Poly-Prep chromatography column</td>
			<td>BioRad</td>
			<td style="width:119px;">7311550</td>
			<td>MS2-affinity purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PstI</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">ER0615</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit 3 Fluorometer</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:119px;">15387293</td>
			<td>RNA quantity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">RNAPro Solution</td>
			<td style="width:187px;">MP Biomedicals</td>
			<td style="width:119px;">6055050</td>
			<td>Mechanical lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">ScriptSeq Complete Kit</td>
			<td style="width:187px;">Illumina</td>
			<td style="width:119px;">BB1224</td>
			<td>Preparation of cDNA librairies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Spectrophotometer Genesys 20</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">11972278</td>
			<td>Bacterial cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">SpeedVac Savant vacuum device</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">DNA120</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Stratalinker UV Crosslinker 1800</td>
			<td style="width:187px;">Stratagene</td>
			<td style="width:119px;">400672</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">T4 DNA ligase</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">EL0014</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">TBE (Tris-Borate-EDTA)</td>
			<td style="width:187px;">Euromedex</td>
			<td style="width:119px;">ET020-C</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">ThermalCycler T100</td>
			<td style="width:187px;">BioRad</td>
			<td style="width:119px;">1861096</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td style="width:187px;">Sigma Aldrich</td>
			<td style="width:119px;">P9416-100ML</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-ray film processor</td>
			<td style="width:187px;">hu.q</td>
			<td style="width:119px;">HQ-350XT</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">X-ray films Super RX-N</td>
			<td style="width:187px;">FujiFilm</td>
			<td style="width:119px;">4741019318</td>
			<td>Northern blot assays</td>
		</tr>
	</tbody>
</table>

ms2-affinity purification coupled with rna sequencing in gram-positive bacteria

虽然小的调节RNA（SRNA）在生命的细菌领域很普遍，但由于难以确定其mRNA目标，其中许多RNA的功能仍然很差。在这里，我们描述了MS2亲和力纯化与RNA测序（MAPS）技术的修改协议，旨在揭示特定sRNA在体内的所有RNA合作伙伴。从广义上讲，MS2 的贴合器与感兴趣的 sRNA 的 5' 四肢融合在一起。然后，此构造以体内表示，允许 MS2-sRNA 与其细胞伙伴进行交互。细菌收获后，细胞被机械地解化。粗提取物被加载到以前涂有MS2蛋白的淀粉样色谱柱中，该色谱柱与麦芽糖结合蛋白融合。这能够对 MS2-sRNA 进行特定捕获并交互 RNA。洗净后，通过高通量RNA测序和随后的生物信息分析确定共同纯化RNA。以下协议已在Gram阳性人类病原体 金黄色葡萄球菌 中实施，原则上可转为任何革兰氏阳性细菌。综上所述，MAPS 技术是深入探索特定 sRNA 监管网络的有效方法，可提供其整个目标图的快照。然而，必须记住，MAPS 确定的假定目标仍需要通过补充实验方法进行验证。

具有代表性的结果源于对金黄色葡萄球菌29的RSAC靶点的研究。RsaC 是一种非常规的 1，116 nt 长 sRNA。其 5' 端包含多个重复区域，而其 3' 端 （544 nt） 在结构上是独立的，并包含与其 mRNA 目标的所有预测交互站点。当锰 （Mn） 稀缺时，就会诱导这种 sRNA 的表达，这通常在宿主免疫反应的背景下遇到。使用 MAPS 技术，我们识别了几个 mRNA 直接与 RsaC 相互作用，揭示了它在氧化?...

Watch this Scientific Journal Video about MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria at JoVE.com

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

MAPS 技术已开发出来，以仔细审查体内特定监管 RNA 的目标。感兴趣的 sRNA 标有 MS2 贴录器，通过 RNA 测序实现其 RNA 合作伙伴的共同纯化及其标识。此修改后的协议特别适合革兰氏阳性细菌。

MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

MS2亲和力纯化与RNA测序或 MAPS 相结合，已开发出来，以识别任何对细菌感兴趣的 RNA 的整个靶点。这项技术使得能够独立于监管机制识别任何类型的 sRNA 合作伙伴。依赖sRNA的监管网络的定性将有助于更好地了解宿主细胞内细菌的毒性因素产生、生物膜形成和生存，并最终对抗葡萄球菌感染。此协议可应用于任何致病性或非致病性克阳性细菌。展示这个程序的将是来自帕斯卡尔·隆比博士实验室的博士生诺米·默西尔。首先，在三毫升的BHI介质中生长一组携带pCN51 P3 MS2 sRNA或pCN51 P3 MS2质粒的菌株，并辅以重复中每微升红霉素10微克。将每个隔夜培养在 50 毫升的新鲜 BHI 介质中稀释，辅以红霉素，达到 0.05 的 OD 600。在 37 摄氏度下培养文化，在 180 RPM 下摇晃 6 小时。将每个培养物转移到一个50毫升的离心管中，在4摄氏度下将管子在2900倍G下离心15分钟，然后丢弃超自然物，在零下80摄氏度下冷冻和储存，或将颗粒留在冰上，直接进行机械细胞裂解。为了进行机械细胞裂解，在5毫升缓冲A中补充颗粒，并将再发射的细胞转移到15毫升离心管，并带有3.5克二氧化硅珠。将管子插入机械单元溶解仪器中，以每秒四米的速度运行 40 秒的循环。将管子在2，900倍G中离心15分钟，然后恢复超母，并将其保持在冰上。确保超自然是明确的，重复离心，如果不是。取出柱尖，然后将色谱柱放入柱架中，用超纯水清洗柱子。加入300微升淀粉样酶树脂，用10毫升缓冲剂A.Dilute 1，200皮卡莫莱MBP-MS2蛋白在6毫升的缓冲A中清洗柱子，并加载到柱中。用 10 毫升缓冲器 A.下一步清洗柱子，执行 MS2 亲和力纯化。将细胞解体加载到柱中，并在干净的收集管中收集流过的分数。用 10 毫升缓冲器 A 清洗柱子三次，收集洗涤分数，然后用一毫升缓冲 E 将柱子稀释，并在两毫升微管中收集洗涤分数。将所有收集的分数保留在冰上，直到RNA提取。使用每个分数的一毫升进行RNA提取。在RNA中加入一卷酚，然后大力混合，然后在20摄氏度下将混合物在16，000倍G中分离10分钟。将上一阶段转移到一个干净的两毫升微管。加入一卷氯仿异酰醇，重复离心，然后将上相转移到新管中。在pH 5.2时加入2.5卷100%冷乙醇和0.1卷三摩尔醋酸钠，在零下20摄氏度下过夜沉淀。第二天，离心机管在16，000倍G和4摄氏度下15分钟。用移液器慢慢去除乙醇，注意不要干扰颗粒。加入500微升80%的冷乙醇和离心机5分钟。通过缓慢管道丢弃乙醇，然后使用真空浓缩器在运行模式下干燥颗粒五分钟。将颗粒在适量的超纯水中补充。此协议用于研究 S.aureus 中的 RsaC 目标。使用 MAPS 技术识别了几个直接与 RsaC 相互作用的 mRNA，揭示了其在氧化应激和金属相关反应中的关键作用。为了确认MS2 sRNA的构造并可视化其表达模式，细胞在BHI介质中经过2、4和6个小时的生长后被收获。提取了RNA，并使用 RsaC 特定的 DIG 探测器进行了北污点分析。内源性 RsaC 水平在生长 6 小时后显著增加。重要的是，MS2-RsaC 544 和 MS2-RsaC 1，116 的水平可与 6 小时内的内源 RsaC 相媲美。进行了亲和力纯化，从野生型菌株中的粗提取物、流经和洗脱分数中提取RNA，仅表达 MS2 标签和表达 MS2-RsaC 544 构造的突变 RsaC 菌株。1，116核苷酸长内源性RSAC在回流部分被丰富，但由于其长度和复杂的次要结构，它与亲和力柱非具体地相互作用。MS2-RsaC 544在浓缩部分高度浓缩，表明它被MS2-MBP融合蛋白成功保留。生物信息学分析显示，根据MS2 sRNA和MS2控制之间的折叠变化，假定mRNA目标，表明草药是第一个假定目标。在 MS2 亲和力纯化后，对北方污点进行了针对 SODA 的 DIG 探头分析，结果表明，与 MS2 控制相比，sodA 与 MS2-RsaC 544 有效共丰富。在尝试此协议时，请记住，添加 MS2 标签会影响 sRNA 确认、稳定性或功能。这一点应仔细监测。地图提供了 sRNA-RNA 目标配对的快照。进一步的实验验证将解开它们的功能。该技术是构建任何细菌 sRNA 监管网络的强大工具。

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

生物素和光裂解链接器所附 rna 对高分子配合物的单步纯化

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

体外和体内实验的位定向诱变-以大肠杆菌rna相互作用为例

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

极快速和特殊的代谢标签的RNA在Vivo与4-Thiouracil （Ers4tU）

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

大鼠胰腺组织快速、经济高效的RNA提取

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

细菌中的RIBO-seq：NGS测序样本收集和库准备协议

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...