Name: quantification of the abundance and charging levels of transfer rnas in escherichia coli
Uploaded: 2017-08-22T14:00:00
Description: Watch this Scientific Journal Video about Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli at JoVE.com

作者感谢玛丽特拉 Warrer 出色的技术援助。这项工作得到丹麦议员支持独立研究中心 |自然科学 [1323年-00343B] 和丹麦国家研究基金会 [DNRF120]。

1.细菌培养制备。
<ol><li>成长所有文化在摇瓶培养/瓶体积比的顶多 1/6。在一个典型的实验设置，成长在 37.0 ° C 文化和摇晃在 160 rpm 在 morpholinepropanesulfonic 酸 （拖把） 最小中型 18 辅以首选的碳源，例如 0.2%的葡萄糖，为至少 10RNA 收获前的连续世代。</li>
	<li>RNA 收获的前一天稀释所需应变的拖把最小介质方式将所需的 OD 436 到达所需的时间第二天穿不了的文化。计算?...

<ol>
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A., Elf, J., Ehrenberg, M., Pan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+charging+of+tRNA+isoacceptors+induced+by+amino-acid+starvation.">Selective charging of tRNA isoacceptors induced by amino-acid starvation.</a> EMBO Rep. 6 (2), 151-157 (2005).</li><li>Zhou, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+reference+genes+for+quantifying+transcriptional+responses+of+Escherichia+coli+to+protein+overexpression+by+quantitative+PCR.">Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR.</a> BMC Mol Biol. 12 (1), 18 (2011).</li><li>Jacobson, A., Gillespie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+events+occurring+during+recovery+from+prolonged+glucose+starvation+in+Escherichia+coli.">Metabolic events occurring during recovery from prolonged glucose starvation in Escherichia coli.</a> J Bacteriol. 95 (3), 1030-1039 (1968).</li><li>McCarthy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+magnesium+starvation+on+the+ribosome+content+of+Escherichia+coli.">The effects of magnesium starvation on the ribosome content of Escherichia coli.</a> Biochim Biophys Acta (BBA)-Specialized Section on Nucleic Acids and Related Subjects. 55 (6), 880-889 (1962).</li><li>Zundel, M. A., Basturea, G. N., Deutscher, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+ribosome+degradation+during+starvation+in+Escherichia+coli.">Initiation of ribosome degradation during starvation in Escherichia coli.</a> Rna. 15 (5), 977-983 (2009).</li><li>Piir, K., Paier, A., Liiv, A., Tenson, T., Maivali, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+degradation+in+growing+bacteria.">Ribosome degradation in growing bacteria.</a> EMBO Rep. 12 (5), 458-462 (2011).</li><li>Schaechter, M., Maaloe, O., Kjeldgaard, N. 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此协议说明如何同时测量特定大肠杆菌的收费水平转运和比较不同样品中转运的相对水平。议定书 》 的关键点是 1） 处理方式保留细胞 tRNA 的收费水平，2） 正常化 tRNA 量方式，可以可靠地比较不同样品中的相对 tRNA 水平，和 3） 确保 sp 样品ecificity 的选定的探头，用于转运的兴趣。这些点在下面分别讨论。
tRNA 净化
转运是纯化大肠杆菌在温?...

在下文中，我们目前的量化具体转运方法和测量他们的收费水平由北弄脏。该方法基于第一次由 Varshney等人的技术1.由成三氯乙酸 (TCA) 收获细胞和保持在 0 ° C，整个 RNA 纯化的样品，tRNA 与氨基酸之间的酯键是保守的2，，34。Aminoacylated 转运可以区分从 nonacylated 同行通过凝胶电泳和北弄脏，适当降低流动性 aminoacylated tRNA 凝胶，引起共价键绑定的氨基酸5中。此外，我们目前的协议规范 tRNA 数量由加法的穗状花序在细胞特异性表达很少使用的 tRNA全 4。
转运是一些最丰富的分子在细菌细胞和翻译机械绝对重要部分。转运绑定氨基酸并将它们传输到翻译的核糖体。氨基酸对转运 （氨或充电） 的绑定被通过氨酰 tRNA 合成酶。不同带电的 Trna 的相对丰度是合成的重要为确保保真度的蛋白质，因为同源任职被控为给定的信使 RNA (mRNA) 密码子 tRNA 增加附近同源 trna 上将的可能性错误地将其氨基酸交付越来越多肽链在核糖体6。TRNA 的重要性反映在大肠杆菌细胞广泛响应 tRNA; 收费水平严重下降严格的响应。严格的反应中转运、 核糖体 Rna 和大多数基因的合成降低有利于与氨基酸生物合成和应力生存相关的特定基因转录和细胞的增长速度大大降低7.此外，最近由我们和其他人的工作表明，大肠杆菌积极降低其 tRNA 翻译4，8的极限应力是响应表明 tRNA 水平调整可能绝大多数为应付这样的压力是重要的。因此，可靠的测量 tRNA 丰度和收费水平将充分了解细菌应激反应的重要工具。
大肠杆菌常用表达重组蛋白和由于密码子用法的物种之间存在差异，次优的表达是一个经常遇到的问题9。这可以由表达式的翻译重组 mRNA10所需的额外转运绕过。收费水平在这种菌株的 tRNA 的测量可以引导疑难解答工作及帮助优化蛋白表达。
此方法还使检测的"mischarging";与一个非同源的氨基酸的 tRNA 分子 aminoacylated。由不同的氨基酸酰化可能会导致 tRNA 要迁移与略有不同的速度通过聚丙烯酰胺凝胶1，11。在某些情况下，该方法也可用于区分不同的修饰模式否则为相同转运3日。
另一种建立生化过程的 tRNA 收费水平调查是高碘酸氧化。这种方法依赖于观察 aminoacylated tRNA 保护从高碘酸钾氧化和不带电的 tRNA 不是。后高碘酸氧化处理充电 tRNA 用于估计收获 RNA 的收费水平。然而，充电几个转运已被证明受治疗从而提供一些不准确12。这里介绍的方法从纯化 RNA 直接充电，因此从化学或酶促反应中排除任何偏见的措施。此方法的一个局限是只有一个 tRNA 种类检测一次，所以虽然同样北污点可以剥离和 reprobed 为多个转运，既费时又有点费力在许多转运上收集数据。
规范样品到彼此的可靠方法是十分重要的研究，目标是跨不同的样品比较分子的相对水平。在这里我们介绍正常化过程为大肠杆菌细胞特异性表达稀有 tRNA全小分装为穗状花序在添加到 RNA 纯化前的所有实验样品的 RNA。此方法很有用不仅审查转运时但相对定量的任何种类的 RNA 时的实验装置是这样没有内源性 RNA 可以信任要出席所有样品中相同的细胞浓度。例如，"管家"RNA 样核糖体 RNA 水平经常用作内源性的参考比较另一个 RNA 之间不同的相对数量样品13。但这是没有多大用处如果参考 RNA 细胞浓度变化之间的样品，可以出现这种情况为核糖体 RNA，如果采样发生在应力响应15，，1617期间或进入固定相17。增加了穗在细胞 RNA 纯化前对实验样品提供规范化的采样设置独立固体和准确的方式。标准化的另一种是另外的一个或多个穗状花序在 Rna 的实验样品 RNA 纯化后。然而，这种方法不占任何在 RNA 恢复样品之间的差异。
利用大肠杆菌细胞 RNA 参考为穗状花序在单元格 （见图 1） 在实验中对大肠杆菌有它结果的缺点，overexpress 另外少量的外源性大肠杆菌的总 RNA样品。我们通过分析示例包含只有穗在细胞中平行实验的样本 （见图 2，车道标记为"全"蛋白免疫印迹北） 更正此添加。议定书 》，提出了一种针对大肠杆菌K-12 但可能是适用于大多数的细菌种类。

<table>
	<tbody>
		<tr>
			<td>Urea</td>
			<td>Merck</td>
			<td>57-13-6</td>
			<td>Purity &#62;99%</td>
		</tr>
		<tr>
			<td>Polyacrylamide</td>
			<td>Serva</td>
			<td>79-06-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis (N,N&#39;-Methylene-bis-acrylamide)</td>
			<td>BIO-RAD</td>
			<td>161-0201</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid (TCA)</td>
			<td>Sigma</td>
			<td>76-03-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Merck</td>
			<td>108-95-2</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Herring Sperm DNA</td>
			<td>Sigma</td>
			<td>100403-24-5</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polivinylpyrrolidone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P32 &#947;ATP</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA oligos (probes)</td>
			<td>TAG Copenhagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybond N+ membrane</td>
			<td>GE Healthcare</td>
			<td>RPN203B</td>
			<td></td>
		</tr>
		<tr>
			<td>Crosslinker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electroblotter</td>
			<td>BIO-RAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000 Scanner</td>
			<td>GE Healthcare</td>
			<td>28955809</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydridization oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Geiger-M&#252;ller tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphor imager screen</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Flasks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 ml centrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
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quantification of the abundance and charging levels of transfer rnas in escherichia coli

转运 RNA (tRNA) 是任何一种生物的平移机构的重要组成部分。转运绑定并将氨基酸转移到翻译的核糖体。不同的 Trna 的相对水平和 aminoacylated 比 tRNA 到总 tRNA，称为收费水平，是在确定的精度和速度的翻译的重要因素。因此，丰度和收费水平的转运是重要的变量来衡量学习蛋白质合成，例如在不同应力条件下的时候。在这里，我们描述为收获 tRNA 和直接测量的相对丰度和特定 tRNA 物种在大肠杆菌中的绝对收费水平的方法。TRNA tRNA 和其氨基酸之间不稳定的纽带保留的一种收获。RNA 然后遭受凝胶电泳和北弄脏，导致分离带电和不带电的转运。具体转运不同样品中的水平可以相比的穗状花序在细胞正常化添加。在 RNA 纯化之前, 我们添加 5%的过量罕见的 tRNA全对每个样本的大肠杆菌细胞。样品中感兴趣的 tRNA 物种的数量然后归的 tRNA全在相同的样本量。穗状花序在细胞 RNA 纯化前的加法有另外它也占任何细胞裂解效率差异样品的纯化穗在 Rna 的优势。

使用过程形容这里，丰度和收费水平的三个转运测定大肠杆菌K-12 之前和期间氨基酸饥饿。
精氨酸营养缺陷型菌株 NF9154 (thr 列伊他啊 thi mtl supE44)了种植至少十代拖把辅以 0.4%的甘油、 50 微克/毫升苏氨酸、 亮氨酸、 精氨酸、 和 5 微克/毫升组氨酸在摇晃在 37 ° C 的最小介质中160 转/分。精氨酸饥...

Watch this Scientific Journal Video about Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli at JoVE.com

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

在这里，我们提出一种直接测量转移 RNA 从纯化的大肠杆菌RNA 以及方式比较的转运 RNA 相对水平或任何其他短的 RNA，跨越不同的样本基础，增设的穗状花序在收费水平方法单元格引用基因的表达。

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Research

JoVE Journal

Biochemistry

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;：设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Quantification of Hypopigmentation Activity In Vitro

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

利用病毒衍生系统在大肠杆菌中大规模生产圆形脚手架上的重组 rna

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

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 ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

使用含有微型基因的Alu元素来分析圆形RNA

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Quantification of Coenzyme A in Cells and Tissues

细胞和组织中辅酶A的定量

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...