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。计算公式用于接种的穿不了区域性的体积: <img alt="Equation 1"src="/files/ftp_upload/56212/56212eq1.jpg"/> 注: 这里，OD 新 表示所需的 OD 436经过孵化，V 新 表示的稀释文化卷，OD 老 表示 OD 436 的穿不了的文化，从被稀释，G 表示的数代人的文化将会成长，从时间 (t 1) 稀释到的时间需要 (t 2) 的第二天，可以计算为: 时间差值 （min) t 1 和 t 2 除以生成时间 （分钟）。为确保稳态生长在采样时，应该是 G &#8805; 10;V 老 表示外种植文化应该转移到新鲜的培养基中的卷。这个方程是一个近似值，不考虑任何滞后期;经常观察后稀释外种植文化在新媒体中的一种现象。
	<ol><li>生长条件将取决于实验的目的但是限制细胞到细胞变异 tRNA 水平，提高重复性的结果，（推荐） 成长文化来稳定前采样状态 19.</li></ol></li></ol> 2。穗状花序在细胞制备 注: 表示编码 tRNA 全 异丙基 β-D-1-thiogalactopyranoside (IPTG) 控制下的 全 基因的 大肠杆菌 菌株 MAS1074，-诱导型启动子用作穗中应变。
<ol><li>摊薄 MAS1074 OD 436 的拖把 0.05 到穿不了文化最小培养基 100 微克/毫升氨苄西林和 0.2%的葡萄糖。成长在 37 ° C，震动在 160 rpm 文化。 
	注: 请参阅 讨论 用于替代参照应变。</li>
	<li>在 OD 436 ~ 0.1，添加 IPTG 至终浓度为 1 毫米，诱导表达的 tRNA 全。长出的文化对 OD 436 ~0.5 (~ 3 4 h 的增长) 和将培养瓶移到冰浴。保持在冰上的穗状花序在细胞培养，直到所有样本的 RNA 纯化都有都收获。</li>
</ol> 3。实验样品收获。
<ol><li>预暖 (37 ° C) 一个上限 100 毫升锥形瓶 （或类似） 包含 4 毫升 10%(w/v) 三氯乙酸 (TCA) 为每个样品放置在洗个温水澡。 
	注: 谨慎，TCA 后加热，分解生产包括氯化氢和氯仿的有毒和腐蚀性烟气。在水中的解决方案是一种强的酸;它与基地剧烈反应，是对许多金属有腐蚀性。使用这种液体时，应采取适当的预防措施。</li>
	<li>前夕细胞收获，测量和记录文化 OD 436。</li>
	<li>收获细胞，4 毫升的培养液转移到预热的烧瓶含有 TCA。</li>
	<li>混合样品通过用手旋转烧瓶，5 美国混合与 TCA 立即禁用所有酶的活性，从而保留转运的收费水平。 
	注意: 如果要收集多个样品，牢记收获的样品 TCA 冰上直到所有样本。</li>
</ol> 4。另外的穗状花序在细胞 注: 在步骤 2 中介绍的穗状花序在细胞的制备.
<ol><li>时所有样品 RNA 制备已收集，每个样品中添加 5%穗在单元格 （基于 OD）。 
	注: 穗在细胞培养所需的卷由公式计算: <img alt="Equation 2"src="/files/ftp_upload/56212/56212eq2.jpg"/> 凡 V 穗 表示卷的穗状花序在细胞培养需要 Vexp表示该卷收获实验细胞培养 （无 TCA)，OD exp 表示 OD 436 实验细胞培养成 TCA 收获时。OD 穗 表示 OD 436 的穗状花序在细胞培养。如果多个样品在不同耗氧物质收获，穗在细胞添加量应根据上面的公式，为每个样本调整。</li>
	<li>量化从穗在细胞贡献 RNA 物种感兴趣，准备只含有穗中细胞与 4 毫升 TCA 混合 4 毫升穗在细胞样品。准备 RNA 从这个示例中相同的方式与其他示例。</li>
	<li>（可选） 采取另一瓶含只给了实验样品，无穗在细胞，并将其量化实验样品给 tRNA 的贡献包括 全 水平; tRNA 全 在 中的内源性水平大肠杆菌 K-12 是可以忽略不计低。</li>
</ol> 5。RNA 制备 注: 此处所述的 RNA 制备协议基本上是由 Varshney 等人 描述 1;要避免在净化过程中的脱乙酰基的 aminoacylated 转运是重要的是保持样品在酸性 ph 值和在整个过程中纯化的 0 ° C。使用清洁无菌管/烧瓶来保存样品和解决方案，并准备所有解决方案与超纯水 （18.2 MΩ·cm 电阻率） 水.
<ol><li>每个样品到冰鲜离心管倒 8 毫升。离心样品 10 分钟在 9,500 x g 4 ° C.完全删除上清液，并在 0.3 毫升冷 0.3 M 醋酸钠，ph 值 4.5 和 10 毫米 EDTA。</li>
	<li>转移到冷 1.5 毫升离心管中每个样品和添加 0.3 毫升苯酚与相同的缓冲区平衡。 
	注: 谨慎，苯酚产生有毒气体和对皮肤高度腐蚀性。</li>
	<li>涡每个样品为 15 10 x s 与至少 1 min 之间每个旋涡。在涡流之间保持样品在 0 ° C.使用频繁的停顿，以确保样品保持冷。</li>
	<li>离心机样品 15 分钟在 20,000 x g 4 ° C.转让水阶段 （上部阶段） 到新的 1.5 mL 离心管。像以前一样添加 0.3 毫升冷苯酚和涡 4 x 15 s.</li>
	<li>离心机以离心 10 分钟 20,000 x g 4 ° C.转让水相向新、 冷 1.5 毫升的微量离心管含 96%乙醇 (~ 750 µ L) 量的 2.5 倍。沉淀 RNA 通过孵化管 1 h 在-20 ° C 或一夜之间在-80 ° C.</li>
	<li>离心机样品 30 分钟在 20,000 x g 4 ° C.弃上清，仔细而不会干扰 RNA 颗粒添加 1 毫升冷 70%乙醇。轻轻地反转一次洗用乙醇管内侧。 
	注: 颗粒很难看到在这一步。</li>
	<li>离心机以离心 10 分钟 20,000 x g 4 ° C.完全删除上清液和风干的颗粒，直到离开没有乙醇。通过大力旋涡中 20 微升冷 10 毫米钠醋酸 ph 值 4.5 和 1 毫米 EDTA 悬小球. 
	注意: 不过度干颗粒，它将变得困难，重悬。</li>
	<li>（可选） 评估测定 RNA 浓度测定吸光度在 260 nm。</li>
	<li>（可选） 通过琼脂糖凝胶电泳 20 评估质量的纯化 RNA。</li>
	<li>商店样品在-80 ° C. 
	注: 议定书 》 能够在这里暂停。</li>
</ol> 6。化学 deacylated 控制样品的制备 注: 区分的 aminoacylated 乐队从 deacylated 对应的北侧 tRNA 污点，化学 deacylated 分装备碱处理。大多数目的而言，这是足够到 deacylate 单一样本的每个北污点。
<ol><li>解冻的 RNA 样本在冰上。转移到新管中的 RNA 样品 4 µ L 分装。添加 46 µ L 三 HCl ph 值 9.0。孵育 2 h 在 37 ° C</li>
	<li>添加 15 微升 0.3 M 醋酸钠的 ph 值为 4.5，随后添加 125 µ L 的 96%的乙醇。沉淀 RNA，在-20 ° C 1 h.</li>
	<li>离心机以离心 30 分钟 20,000 x g 4 ° C.完全除去上清，悬在 4 µ L 冷 10 毫米钠醋酸 ph 值 4.5 和 1 毫米 EDTA。 
	注: 议定书 》 可以暂停在这里，如果样品被存放在-80 ° C.</li>
</ol> 7。凝胶电泳和北弄脏 <ol><li>混合 4 µ L 样品 (未经处理或化学 deacylated) 与 6 µ L 加载缓冲区 （0.1 M 醋酸钠 (pH 5.0)、 8m 尿素、 0.05%溴酚蓝和 0.05%二甲苯蓝）。 
	注: 请记住，包括样品只有穗中的单元格，化学 deacylated 样品 （和无穗在细胞，如果一个人准备样品）。</li>
	<li>装载到 0.4 m m 厚 6.5%聚丙烯酰胺凝胶 (19:1 acrylam-ide:bisacrylamide) 包含在 0.1 M 醋酸钠 (pH 5.0) 8m 尿素样品。60 厘米长凝胶用于适当分离的 tRNA 物种。</li>
	<li>在 4 ° c，10 V/cm 凝胶电泳法分离 RNA 直到溴酚蓝到达的凝胶 (~ 20 h) 底部。</li>
	<li>地区从二甲苯蓝染料和 20 厘米下来对溴酚蓝染料用于印迹 （两种染料之间的距离应该是 25-30 厘米）。仔细单独两个玻璃板材，以便这种凝胶仍然是其中之一。一块薄薄的滤纸现降至所需的吸墨纸区域的大小，放在将用于吸水凝胶的一部分。凝胶中未涵盖的过滤纸的部分是切断和丢弃。 使用过滤纸小心地提起凝胶这块远离玻璃板。 
	注: 大凝胶是很脆弱所以小心轻放。</li>
	<li>正电荷的尼龙膜 （例如中 N + 膜） 放在凝胶上，是在使用 40 毫米醋酸 (pH 8.1)，2 毫米 EDTA 作为传输缓冲区的 90 分钟 20 V 裁。</li>
	<li>交联 RNA 对使用 UV 光膜 (0.12 J/厘米 2). 
	注: 膜现在可以存储在室温和议定书 》 能够在这里暂停。</li>
</ol> 8。探针的设计与验证 注: 小心北污点探针的设计与验证至关重要获得可靠的结果，避免与其他 RNA 种类不需要的交叉杂交。
<ol><li>使用短合成寡核苷酸探针。设计中一种方式，它是相辅相成的感兴趣的 tRNA 序列最独特的地方 — — 这往往是反密码子环的探针序列。 
	注: 从各种生物的预测 tRNA 序列的列表可以找到在基因组的 tRNA 数据库 (GtRNAdb) 21.</li>
	<li>要获得预测的序列的长度调整熔化温度 55-65 ° c。</li>
	<li>爆炸 （基本局部比对搜索工具） 所选序列对基因组序列的菌株在实验中，用来验证序列是唯一选定的 tRNA， 22 中所述。 
	注: 表 1 列出了建议的探针序列，为几个不同的 Trna 的 大肠杆菌 K12。如果探针是特定为感兴趣的 tRNA，只有两个波段上均可见北污点 (带电和不带电的 tRNA) 和只有一个乐队是在车道与 deacylated 样本 (不带电 tRNA) 中可见。如果两个以上乐队目前或如果需要进一步验证的特异性探针，见讨论。</li>
</ol> 9。北部的印迹杂交 注: 在以下步骤中，膜按顺序杂交探针补充所需的特定转运，包括探头特定参考 tRNA 全。
<ol><li>膜置于杂交管中，加入 6 毫升杂交溶液 (0.9 M 氯化钠，0.05 M NaH 2 PO 4 (pH 7.7)，5 毫米 EDTA，0.5%(w/v) SDS，100 毫克/毫升的剪切和变性鲱鱼精子 DNA 和 5 x Denhardt &#39; s 解决方案 (100 xDenhardt &#39; s 解决方案 = 2%牛血清白蛋白、 2%吡咯烷酮 40 和 2%蔗糖))。</li>
	<li>预杂交膜中的缓冲区的旋转在 42 ° C.添加 30 皮摩尔放射性寡聚 DNA 探针 5 1 h &#39; 端-带有 32 P （编写由山姆布鲁克 等人 所述 23) 注: 警告，高能 β 排放量 32 P 可以呈现大量的皮肤和眼睛剂量危害。当使用 32 页放射性废物应按处理适当的体制安全协议时应采取适当的预防措施。</li>
	<li>孵育探针与膜隔夜旋转在 42 ° C.删除探针一次性 10 毫升吸管。 
	注意: 如果储存在冰箱的探针可重复使用。</li>
	<li>在杂交管中，洗膜不久在 50 毫升的 0.3 M 氯化钠，柠檬酸钠 30 毫米，0.1%(w/v) SDS 在室温。 
	注意: 这第一次洗包含多绑定的探针和必须作为高度放射性处理。</li>
	<li>重复一次，一步 9.4，然后将膜移动到平塑料集装箱或托盘包括盖子。覆盖膜与洗涤液 （0.3 M 氯化钠，柠檬酸钠 30 毫米，0.1%(w/v) SDS） 和孵育 30min 室温。</li>
	<li>改变洗涤液和持续冲洗，直到令人满意的信号的信噪比得到了 （通常 3-4 更改的洗涤液）。使用盖革-米勒管来检测噪声对信号比通过膜的从一个空的区域辐射测量的更改和比作的地区地方探头具有杂交特指 tRNA。</li>
	为了能够带从膜探针接触之后，<li>保证，（重要） 膜也不干溶出过程之前。将膜放在薄薄的塑料袋或包裹它紧紧地使用塑料包装和密封就是气密的磁带或焊接。在 phosphorimaging 屏幕上放置密封的膜。 
	注: phosphorimaging 屏幕上所需的曝光时间测定探针的具体活动的 RNA 量探讨佛r 和探针，和可以杂交效率相差很大;从几分钟到几天。与经验，在盖革-米勒管膜上检测到的辐射强度给需要的曝光时间很好的说明。为可靠地进行量化，很重要的屏幕不过度曝光。</li>
	<li>Phosphorimaging 屏幕使用激光扫描仪扫描和保存在文件 &#34;.gel &#34; 格式。 
	注: 高信号的信噪比是必要的可靠的量化。探测的 tRNA 应导致在两个频段;一个包含 aminoacylated tRNA （慢迁移） 和一个包含 deacylated tRNA （快迁移）。请参见 图 2。</li>
</ol> 10。剥膜 注: 膜可探测的另一个 tRNA 之前，先前的探头必须先删除由剥膜。
<ol><li>热足够剥缓冲区 （15 毫米氯化钠，柠檬酸钠 1.5 毫米，0.1%(w/v) SDS） 覆盖膜和倒膜。孵育 15 分钟在室温。</li>
	<li>可以用盖革-米勒管检测重复步骤 10.1 直到无辐射; 膜现在可以重新杂交后第九条中所述的步骤。</li>
</ol> 11。量化 tRNA 和收费水平 <ol><li>负载.gel 文件到成像软件 （见 表的材料）。画一条垂直线沿着每个样品车道方式它横跨了大部份的车道的宽度，但不包括乐队的边缘其中信号可以是不平衡的如 图 3A 所示。</li>
	<li>可视化计数从每个车道通过单击 &#34; 分析 &#34;-&#62; &#34; 创建图 &#34;。手动定义表示带电和不带电的 tRNA 从每个车道，两座山峰，如 图 3B 所示。</li>
	<li>从每个峰获得计数，通过单击 &#34; 分析 &#34;-&#62; &#34; 地区报告 &#34;，并记录下每个两个峰的面积。</li>
	<li>当 tRNA 全 和至少一个 tRNA 的兴趣，探讨了污点正常化的 tRNA tRNA 全 感兴趣的水平。
	<ol><li>计算的 tRNA 源自每个车道的穗状花序在细胞比例和从该行车线行驶，利用方程的总信号中减去: <img alt="Equation 3"src="/files/ftp_upload/56212/56212eq3.jpg"/> 注: 在这里，样品 tRNA = 从单线的探测所需的 tRNA; 膜得到的信号样品 全 = 从同一行车线探测 tRNA 全; 对同一种膜得到的信号Ref tRNA = 从车道只有穗在细胞 rna 的同一种膜有人探测其是否需 tRNA; 得到的信号Ref 全 = 从车道只有穗在细胞 rna 的同一种膜有人探测其是否 tRNA 全 得到的信号。</li>
		<li>实现正常化，来自每个车道的更正的 tRNA 值除以来自同一行车线的 tRNA 全 信号。 
		注: 从采样细胞 tRNA 全 信号的贡献是可以忽略不计 (见 图 2，车道标记 &#34;-全 &#34;)。</li>
	</ol></li> <li>计算收费水平每条车道除以信号的峰值代表 tRNA 与信号总和从这两个峰的 tRNA （带电 + 不带电荷）。</li></ol>

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F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (1989).</li><li>Sorensen, M. A., Jensen, K. F., Pedersen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+concentrations+of+ppGpp+decrease+the+RNA+chain+growth+rate.+Implications+for+protein+synthesis+and+translational+fidelity+during+amino+acid+starvation+in+Escherichia+coli.">High concentrations of ppGpp decrease the RNA chain growth rate. Implications for protein synthesis and translational fidelity during amino acid starvation in Escherichia coli.</a> J Mol Biol. 236 (2), 441-454 (1994).</li><li>Tian, C., 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=Rapid+Curtailing+of+the+Stringent+Response+by+Toxin-Antitoxin+Module-Encoded+mRNases.">Rapid Curtailing of the Stringent Response by Toxin-Antitoxin Module-Encoded mRNases.</a> J Bacteriol. 198 (14), 1918-1926 (2016).</li></ol>

作者没有透露。

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和的方式，优化可保留其细胞的收费水平。它是我们的经验本议定书主要提取 Rna 少于 150 台币，因而 tRNA 构成纯化 RNA 的很大一部分。出于此原因，不推荐使用此协议来净化从大肠杆菌的总 RNA。这里介绍的议定书 》 也应适用于检测和定量的其他 Rna 等小分子 RNA (sRNA) 没有任何进一步的调整。因为 RNA 提取协议丰富了短的 RNA 序列它也应该找到使用，即使根据调查 sRNA 低表达。
穗状花序在细胞正常化
比较不同样品 tRNA 的水平，就必须恢复正常水平的是目前在每个细胞都是样品，在相同的平均数量为纠正的 RNA 样本样本变化的成绩单 tRNA 水平回收和凝胶装载。为此目的，我们穗样品与 5%的过度表达稀有 tRNA全在 RNA 纯化前的引用单元格。穗状花序在方法优于使用内源性 RNA 作为参考，因为它允许跨条件没有内源性 Rna 已知与确定性将被发现在同一细胞浓度的 tRNA 水平的比较。利用大肠杆菌细胞作为穗中有缺点，5%的额外大肠杆菌RNA 被添加到所有实验的样品，导致信号的 RNA 感兴趣的地方。这种偏见被占了包括示例包含只的穗状花序在单元格上每个北的污点，和纠正 RNA 值步骤 11.4.1 中所述。表达的 tRNA全在野生型细胞是如此之低，相比其他转运 （见图 2），它并不在量化的显著差异，因而可以忽略不计。不过，在参考菌株 MAS1074，在那里全表示 T7启动子高拷贝质粒，我们估计至少 1000 倍更多 tRNA全驻留 1 mM IPTG 产生比产生的丙肝E.大肠杆菌K-12 在任何情况下。MAS1074 停止成长后两代人的充分 IPTG 诱导。在这一点上，感应 tRNA全中文化的差异很小，并不关心本议定书作为所有整除数的穗状花序在细胞的单个实验取自同一诱导培养的 MAS1074。从其 RNA 杂交探针将没有交叉作用的有机体的细胞也可以作为 MAS1074 的替代方法，增加穗在细胞等用途。硫化叶菌麦芽寡糖细胞已经成功地被用作穗在大肠杆菌tRNA4的实验。然而，大肠杆菌穗在细胞是可能更准确地反映实验的细胞，细胞裂解的程度的因为它是不确定是否美国麦芽寡糖溶解以同样的效率作为大肠杆菌。此外，增长美国麦芽寡糖是更乏味，他们成长在 85 ° C ~ 16 h 代时间。
确保所选的转运的特异性
如果 aminoacylated 和 deacylated tRNA 已成功应北方膜检测纯化后的两个乐队。此两种频带之间的距离会有所不同根据调查，具体 tRNA 和是在大小、 极地属性和特定的氨基酸导致如果它绑定到特定的 tRNA5 的构象差异合并的结果.例如，aminoacylated 和 deacylated 之间的距离 tRNA 大于为 tRNAargVYZQ它是 tRNAgltTUVW，如图 2所示。由于这里用于分隔 RNA 凝胶的大大小，分辨率也很高要区分和不带电的 tRNA 还要区分其差别，在序列的长度，从彼此不同的 Trna 绝大多数组成和修改模式。亮氨酸市场接受转运2所示，可分为甚至 isoaccepting 转运。然而，请注意，在这项研究是不可能区分 tRNA紊流，tRNAleuPQV作为序列中的差异是只有一个 G T 替代。
如果两个以上的乐队出现在北部膜上可能的交叉反应与其他 Rna 探针的后果。通过提高温度增加的洗涤步骤 （步骤 9.5） 紧缩通常可以解决这个问题。在 55 ° C 洗 30 分钟通常是足够的。如果增加洗紧缩不会删除交叉杂交，解决办法可以是设计新的探针是相辅相成的 tRNA 序列不同 （通常部分重叠） 部分。其他乐队也可以从以前的探头，由于不足剥离膜的结转。要防止这种情况，通过重新探索之前暴露膜做地带控制。如果乐队出现在控制扫描膜可能需要额外的剥离。它通常是可能的重新探讨膜至少 8-10 倍，但后几个探头可以很难完全剥离。如果是这样，膜可以存放几个月前重新探讨作为32P 衰变快。
进一步核查的特异性探针可以通过执行北与细胞一组条件下收获利益的誊本预计将在可预见的方式影响的污点。例如，从一个质粒或为同源的氨基酸，氨基酸饥饿 tRNA 的诱导表达应导致增强相对表达水平或更低的收费水平，分别，trna 上感兴趣。

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>
	</tbody>
</table>

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。精氨酸饥饿介绍了文化和再悬浮在新鲜拖把培养基作为以上但没有精氨酸的过滤。精氨酸饥饿，蛋白质合成强烈减少了，但仍然在使用精氨酸发表的现有蛋白质周转的低水平。样品在收获时间点显示在图 4 中。所有样品都收获到 TCA 后，5%的超量表达 tRNA全，穗状花序在细胞 MAS1074 添加到每个样本中所示图 1。RNA 是从样品分离纯化、 由电泳法，分离和涂抹到膜上，如议定书 》 中所述。膜被探测 tRNAargVYZQ、 tRNAgltTUVW、 tRNAhisR和 tRNA全，如图 2所示。从每个车道的辐射信号量化为每个作为插图在图 3中，更正和归一化，第 11 条所述的四探针。在图 4 中介绍了结果。图 4A显示的所有测试的 tRNA 物种水平迅速降低期间氨基酸饥饿，作为最近报道4。图 4B表明 tRNA 收费水平像预期的那样: 精氨酸接受 tRNA 的带电的分数迅速下降除去其中精氨酸从生长介质，而其他转运的收费水平略有增加饿死因为被控转运积累他们的 decharging 在核糖体率减少由于缺乏底物蛋白合成4时。进一步进入饥饿时期，带电的 tRNA 分数降低到大约前饥饿水平由于绝大多数 tRNA 池 (图 4A)，导致更大的率的其余转运在 decharging 的降解核糖体。相比之下，tRNAargVYZQ的带电的分数增加至少两个原因;1） 激活的严格的响应是指 mRNA，不收取 tRNAarg，成为翻译的限制因素和 tRNAarg是 2) 总池减少 (图 4A) 所以总 tRNA大分数 arg可以通过相同的少量的精氨酸被控。本实验演示如何 tRNA 丰度和 tRNA 收费水平的同时测量提供了高质量的信息，对细胞内蛋白质合成的条件。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56212/56212fig1.jpg"> 
图 1。另外的穗状花序在细胞样品。在此示例中，从单一文化的大肠杆菌NF915 样品的收获时间序列中之前和期间氨基酸饥饿。所示的 NF915 的生长曲线示意图，其中的蓝色小点表示样本收获的点。Y 轴是对数刻度。样品的实验文化收获第 3 节中所述。穗状花序在应变 MAS1074 生长曲线示意图也显示，与样品收获表明作为一个蓝点的点。图的点是为了说明所有实验样品 （在本示例中的大肠杆菌NF915) 收到相同参考文化的穗状花序在细胞分装。对每个样本中，5%的穗状花序在细胞添加到实验细胞，计算基于 OD （见第 4 节），细胞培养物。随后，RNA 是从所有样品分离纯化和用于北方的污点。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56212/56212fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56212/56212fig2.jpg"> 
图 2。北部的一个污点荧光粉成像仪扫描探测表明转运。膜内, 含有 RNA 从大肠杆菌菌株 NF915 收获在指定的时间点之前和之后感应精氨酸饥饿 （分钟）。此外，膜还包含的示例省略了穗在细胞两条行车线 (-全 DA 和-全)，和含有 RNA 从穗在车道细胞唯一 （全）。-全大样品化学包含 deacylated tRNA。代表的带电和不带电的 tRNA 物种的两个带区明确分开。请注意穗在单元格添加没有来自 tRNA全在车道的可以忽略不计的信号。相同的膜是先后剥离和 reprobed 为转运表示在左边。中上部的污点的盒装的区域指示的污点，为后续的 tRNA 探针显示的部分。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56212/56212fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56212/56212fig3v2.jpg"> 
图 3。量化的北污点。tRNA 丰度测量使用软件。(A) A 单线从北污点有人探测其 tRNAargVYZQ的图 2所示。上下箭头指示 aminoacylated tRNA 和 deacylated tRNA，分别。运行与行车线平行的红线添加和用来定义区域为量化。(B) 在 (A) 所示的两个断红线内信号的量化描述作为一个图形，在水平轴表示的距离 （以像素为单位），在 (A) 行的顶部和垂直轴显示的信号强度 （计数）。二山峰包含信号源自从 aminoacylated tRNA 和 deacylated tRNA，分别是手动定义。出口作进一步分析的数据是根据每个两峰区对应的值。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56212/56212fig3v2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56212/56212fig4.jpg"> 
图 4。代表结果: 量化的 tRNA 丰度d 充电精氨酸饥饿时的水平。在图 2中所示的北污点乐队的量化。(A) 相对丰富的 tRNAgltTUVW （红色）、 tRNAargVYZQ （蓝色） 和 tRNAhisR （绿色）。发现在零分钟 （收获前发病的饥饿的样本） 的级别设置为 1。(B) 收费水平的 (A) 在相同的三个转运。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56212/56212fig4large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>探针的特异性</td>
			<td>探针序列</td>
		</tr>
		<tr>
			<td>tRNAargVYZQ</td>
			<td>5'-TCCGACCGCTCGGTTCGTAGC</td>
		</tr>
		<tr>
			<td>tRNAgluTUVW</td>
			<td>5'-CCTGTTACCGCCGTGAAAGGG</td>
		</tr>
		<tr>
			<td>tRNAhisR</td>
			<td>5'-CACGACAACTGGAATCACAATCC</td>
		</tr>
		<tr>
			<td>tRNAleuPQVT</td>
			<td>5'-GTAAGGACACTAACACCTGAAGC</td>
		</tr>
		<tr>
			<td>tRNAleuU</td>
			<td>5'-TATTGGGCACTACCACCTCAAGG</td>
		</tr>
		<tr>
			<td>tRNAleuW</td>
			<td>5'-CTTGCGGCGCCAGAACCTAAATC</td>
		</tr>
		<tr>
			<td>tRNAleuX</td>
			<td>5'-TATTTCTACGGTTGATTTTGAA</td>
		</tr>
		<tr>
			<td>tRNAleuZ</td>
			<td>5'-AAAATCCCTCGGCGTTCGCGCT</td>
		</tr>
		<tr>
			<td>tRNAlysQTVWYZ</td>
			<td>5'-TGCGACCAATTGATTAAAAGTCAAC</td>
		</tr>
		<tr>
			<td>tRNAthrV</td>
			<td>5'-TGGGGACCTCACCCTTACCAA</td>
		</tr>
		<tr>
			<td>tRNAtyrTV</td>
			<td>5'-TCGAACCTTCGAAGTCGATGA</td>
		</tr>
		<tr>
			<td>tRNAselC</td>
			<td>5'-ATTTGAAGTCCAGCCGCC</td>
		</tr>
	</tbody>
</table>
表 1。表的选定转运中大肠杆菌K-12.建议的探针序列

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. ...

quantification-abundance-charging-levels-transfer-rnas-escherichia

Nanyang Technological University

Research

JoVE Journal

Biochemistry

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

9.2K 次观看.  University of Copenhagen. 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。

视频: 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

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

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

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

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

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

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

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

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

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

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

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

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

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

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 is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

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

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

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

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 upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

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 ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Caenorhabditis elegans中 2-阿拉希多诺诺基甘油定量的分解标准合成

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

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

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

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

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

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 Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

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. ...