我们感谢安妮·德容和西热Holsappel的帮助，DNA微阵列幻灯片制作。安妮·德容的生物信息学分析的支持表示赞赏。我们也感谢耶勒Slager审查的文件。穆罕默德·阿夫扎尔和伊尔凡曼苏尔是由GC大学，费萨拉巴德，巴基斯坦在巴基斯坦HEC的师资队伍建设项目的支持。

1.制备媒体，以及细胞培养<ol><li>成长S.如先前所描述的11 肺炎 D39野生型菌株21。接种储存在-80℃下在10％的甘油（以1/100的比例在50毫升无菌试管中）的50ml化学定义的培养基（CDM）为6.4 15最终pH的细胞，但是从氨基酸省略L-丝氨酸混合。 注:两个不同的是存款机使用;一种含含L-丝氨酸（10毫米）的最大浓度的L-丝氨酸（150μM），而另一个的最小浓度。该最小浓度基本上是L-丝氨酸在人血浆中的浓度，目的是比较S的基因表达这两个条件下，肺炎 D39应变。 </li></ol> 2.提取总RNA <ol><li>物料<ol><li>使用酸性酚，核糖核酸级总RNA的分离。 </li><li> Macaloid: <ol><li>暂停2克macaloid在100毫升TE煮5分钟。冷却至RT，并声处理直至macaloid gels.Centrifuge在50毫升的TE pH为8，并储存在4℃下将溶液在2000×g下5分钟，在室温，重悬。 </li></ol></li><li>解决方案: <ol><li>治疗用焦碳酸二乙酯（DEPC）所有的解决方案。添加100微升DEPC / 100ml的溶液，孵育O / N在37℃和高压釜15分钟。 </li></ol></li></ol></li><li>方法<ol><li>增长50毫升S.在50毫升管肺炎 D39细胞培养在37℃（无抖动），直到指数中期阶段（OD〜0.3）。离心培养物在4℃下10,000×g下 2分钟。丢弃媒体并立即冷冻在液氮中的细胞沉淀。 </li><li>预混物加入300μl氯仿:IAA（24:1）和300μl的苯酚（酸苯酚，RNA级）。用500微升的有机相在步骤2.2.3。 </li><li>预先在无RNA螺旋盖管中制备下列混合物:00.5克玻璃珠，50微升10％SDS，500微升酚/氯仿:IAA（如制备步骤2.2.2），macaloid层（150-175微升，不准确的，因为它是非常粘稠）。悬浮于400μl的TE（DEPC）的细胞沉淀并加入再悬浮细胞进入螺旋帽试管中。 </li><li>以破碎细胞，将螺旋帽试管中一个珠打浆机为2×60"的脉冲（&#39;均质&#39;）用1分钟的时间间隔在冰上。离心机的样品在10000×g下（4℃）10分钟。 </li><li>上层相转移至新管中，加入500μl氯仿:IAA（24:1）和离心5分钟，以10,000×g下（4℃）。 </li><li>转移500μl的上层相的至新管中，添加的裂解/结合缓冲液2倍体积（1ml）中，并通过上下抽吸混合。分离总RNA使用RNA分离试剂盒，并按照制造商推荐的协议。 </li></ol></li></ol> 3. R​​NA清理 &lt;<html> OL&gt; <li>从总RNA去除DNA的污染，加上100微升DNA酶I混合（90微升DNA酶缓冲和10μLDNA酶I）和孵育20-30分钟，在15-25ºC。 </li><li>使用RNA酶试剂盒洗干净RNA。获得50微升洗脱体积。 </li></ol> RNA 4.分析<ol><li>确定的分光光度计RNA的浓度。通过使用分析如下（ 图2）确定RNA的质量。 <ol><li>稀释1μl的样品用50微升DEPC水来获得20-200纳克/微升的浓度。 </li><li>使用1微升稀释的RNA试样根据制造商的说明来检查质量上的生物分析仪。 </li></ol></li><li>注:23S的比例约为2.0 16S被认为是好的1 A260单位RNA相当于40微克/毫升。推荐的量标注为10-20微克。 </li></ol> 5. cDNA的制备和标签ntent"&gt;注:以下协议之后进行的cDNA的准备和标签。 <ol><li>退火<ol><li>进行退火反应在300微升的PCR管，保持总RNA 10-15微克的浓度为自制幻灯片或5微克为公司制造的幻灯片。混合的RNA与2微升随机九（1.6微克/微升），并添加无核酸酶水，如果必要，以保持退火混合物的终体积为18微升。 </li><li>保持在退火混合物在70℃下5分钟。此后，冷却该混合物至室温10分钟（退火）和如果需要的话，降速反应到管的底部。将反应管在冰上至少1分钟。 </li></ol></li><li>反转录<ol><li>准备逆转录酶结构如下:18微升退火混合，加入12微升主结构（包括6微升5X第一链缓冲液，3微升0.1M的DDT，1.2微升25X AA-的dUTP /核苷酸混合物和1.8微升倒Transcriptase（1微升的公司制造的幻灯片）。保持反应混合物为2-16小时，在42℃。 </li></ol></li></ol> 6.降解mRNA和cDNA的纯化<ol><li>以从反应混合物中降低的mRNA，加入3微升的2.5M NaOH和地点，在37℃下进行约15分钟。接着，加入15微升2M HEPES游离酸，以中和氢氧化钠。 </li><li>通过PCR清理列净化的cDNA混合，并按照制造商的协议。 </li></ol> 7.测量基因浓度<ol><li>衡量一个分光光度计的cDNA浓度。要继续使用标签，检查的cDNA的浓度至少为60纳克/微升（自制幻灯片）或20毫微克/μL（公司制作的幻灯片）。 </li></ol> 8.标签的cDNA与胺活性染料与纯化<ol><li>使用胺反应性染料来标记的cDNA。直接混合的cDNA用的一个等分试样（5微升）胺反应性染料。 </li><li>在RT孵育该混合物中，在黑暗中，60至90分钟，并直接进行染料标记的cDNA的纯化。通过使用PCR净化柱并按照生产商的方案纯化染料标记的cDNA。在50μl的洗脱缓冲液洗脱的cDNA。 </li></ol> 9.测量标记的cDNA的<ol><li>使用分光光度计来测量的胺反应性染料入的cDNA掺入。检查的胺 - 反应性染料的浓度为至少0.5皮摩尔/微升在50微升的总体积。 </li></ol> 10.混合标记的基因样品<ol><li>自制幻灯片，使用所有的标记cDNA杂交在cDNA的浓度不大于30％的差异。对于公司制造的幻灯片，使用基因与不低于基因浓度为2倍的差异等等。 注意:通常情况下，约300纳克的cDNA，需要对公司制造的幻灯片，相比于REQ这是非常小uired cDNA的量自制的幻灯片。 </li></ol> 11.杂交和洗涤<ol><li>使用以下试剂:半幅水，乙醇99％，SHY缓冲（自制;用40微升酵母RNA）。准备最多1毫升害羞。 </li><li>设备和解决方案的准备<ol><li>干燥前打开真空浓缩和加热器至少1小时。接通杂交炉，用调整到正确的杂交温度设定点（对于肺炎链球菌 DNA微阵列，使用45℃）。同样，预热杂交录像带和烤箱30-60分钟。 </li><li>预热SHY缓冲器在68℃至少30分钟。 </li></ol></li><li>样品制备（标记的cDNA） <ol><li>结合等量标记的cDNA（最多30％差）。使用真空浓缩，在高温（约40分钟），直到体积小于7微升干燥样品。 </li></ol></li><li>升降滑<ol><li>用干净的升降杆单。注意:±30微升可以在滑动加载。 </li><li>清洁挺杆-卡瓦用肥皂，大量的自来水和100％的乙醇。注:脏升降机单给予很高的背景。 </li><li>空气干燥升降，单用气手枪吹走灰尘颗粒。广场上，在两侧的白色聚四氟乙烯衬里朝下幻灯片干净的升降滑。 </li></ol></li><li>加入杂交缓冲液（以标记的cDNA） <ol><li>溶解在干燥的染料样品在7微升的H 2 O和孵育在94℃进行2分钟。 </li><li>随即，加入35微升预热SHY缓冲液（68°C），轻轻混匀，在最大速度为1分钟摆脱沉淀。预热探针在68℃，大约5分钟，直到加载。 </li></ol></li><li>上滑升降滑装配和预热。 <ol><li>将热块的杂交幻灯片固定在50℃。将DNA微阵列载玻片上加热杂交滑座和P的升降滑再热与升降滑了一分钟的幻灯片。执行下面的步骤，尽快。 </li><li>添加40微升样品靶到滑动的末端。允许流体通过毛细力在玻璃表面之间流动。缓慢而仔细地执行所有的移液。 </li><li>保持与升降滑水平的幻灯片在任何时候，慢慢地移动，确保了升降滑不动了。 </li><li>就拿预热杂交纸盒拉出杂交炉并关闭机器。代替滤纸浸泡用3ml 2×SSC（标准柠檬酸盐）中的杂交盒。 </li><li>轻轻将杂交滑套与杂交录像带的幻灯片。关闭杂交录像带，并把在杂交炉试（约16-18小时）。 </li></ol></li><li>滑洗<ol><li>制备新鲜洗涤缓冲液I，II和III（每次洗涤步骤750毫升）中。对于500毫升洗缓冲I，用2×SSC / 0.5％SDS。对于500毫升洗的buff呃II，使用1×SSC / 0.25％SDS。对于500毫升洗缓冲III，使用1×SSC / 0.1％SDS（可选） </li><li>将洗涤缓冲剂在30℃下（以确保SDS溶解）。 </li><li>淹没的幻灯片尽快，但很平缓，在Falcon管填充用50ml洗涤缓冲液I，直到玻璃靠在管的圆锥形底部。 </li><li>几秒钟后，当升降器滑汇到管的底部，取出用镊子滑动而不刮伤阵列和放入所述洗涤台的齿条之后。继续没有时间差的清洗。 </li><li>为5分钟，在500洗载玻片毫升洗涤缓冲液I（同一个清洗站）。给它的第二洗涤20-30分钟在500毫升洗涤缓冲液II（在洗涤站）。同样，在500洗幻灯片5分钟毫升洗缓冲III（可选）（在清洗站）。 </li><li>干燥载玻片2分钟以2000rpm。 </li></ol></li></ol> 12.微阵列分析<ol><li>扫描图像在扫描仪的各波长和保存文件夹"朱庇特工程"。 </li><li>使用软件最初分析扫描文件如前所述22.运行该软件后，选择"打开图像"选项卡中的红色映像文件（-.550）加载为"红色"和绿色的图像文件（-.635）作为"绿色"。通过选择"加载数组列表"选项卡22载有图像文件肺炎链球菌数组列表中的文件加仑（.gal）。 注:此数组列表包括48格的，对每个网格有16列和15列。在网格上每个点代表一个单独的基因和现货信息，包括基因名是通过现货描述文件添加。当场号码从左边给出向右，从上到下。 </li><li>经过仔细察觉的网格，选择选项卡"查找阵列，查找模块，对齐功能"对齐点。对准所有功能后，分析图像通过选择"分析"TAB。创建具有效果，直方图和散射图的新文件。保存从标签文件"保存结果"作为.gpr文件作进一步的分析。 </li><li>所描述的9与内部开发Microprep软件包进行进一步正常化和数据处理。 </li><li>使用独立的生物复制的DNA微阵列数据这是染料交换。执行CyberT实施T-test1的一个变种，并计算假发现率（一级亲属）所描述的9。 </li><li>差异表达基因，以P &lt;0.001和FDR &lt;0.05为标准。 </li><li>上传NCBI上提交页面DNA微阵列数据得到GEO（基因表达总括）登录号。 </li></ol>

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P., Kok, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PePPER,+a+webserver+for+prediction+of+prokaryote+promoter+elements+and+regulons.">PePPER, a webserver for prediction of prokaryote promoter elements and regulons.</a> BMC Genomics. 13, 229 (2012).</li><li>Lanie, J. A., 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=Genome+sequence+of+Avery's+virulent+serotype+2+strain+D39+of+Streptococcus+pneumoniae+and+comparison+with+that+of+unencapsulated+laboratory+strain+R6.">Genome sequence of Avery's virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6.</a> J. Bacteriol. 189, 38-51 (2007).</li><li>Molecular Devices, Corp. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GenePix Pro 6.0 Microarray Acquisition and Analysis Software for GenePix Microarray Scanners-Use's Guide and Tutorial. , (2005).</li></ol>

作者宣称，他们有没有竞争的财务权益。

我们描述了可应用到执行的细菌的全转录组分析一个用户友好的协议。有关此特定技术的关键点是，在其下收获细胞的情况会有所不同。后收获细胞并分离RNA，该技术变得等于对所有类型的细菌的样品，并遵循完全相同的步骤，因此，可以适用于任何类型的细菌培养物。该协议是很简单的，方便的，并开始从RNA分离。我们的RNA分离协议（使用Macaloid和RNA分离试剂盒）是时间效益，因为相比于传统?...

整套的mRNA的丰度（转录组）的在特定时间或在特定条件下的编码通过单细胞生物体或真核细胞的基因组中，包括基因过表达或敲除的研究中，被称为转录。转录使我们能够观察到什么程度的基因都在一个时间点X的特定条件下进行表达，并给了我们如何强烈的基因表达相对于参考信息。 微阵列是在固体基材上的二维阵列（通常是载玻片或硅薄膜电池），它可以被用于使用高通量筛选以测定大量的生物材料，和小型化，复用和并行处理和检测方法。芯片有各种型号，包括DNA，基因芯片，蛋白质芯片，芯片肽，组织芯片，​​抗体芯片，细胞芯片等。 DNA微阵列是基本上微观DNA斑点的组件固定在固体表面上，通常是玻璃。 DNA微阵列被用于测量的基因的表达水平或一组基因的同时或基因型基因组2,3的多个区域。探针的皮摩尔（10 -12摩尔）的每一个DNA斑点代表特定的DNA序列，也被称为记者中存在。从样品中所述标记的mRNA分子被称为"目标"。荧光团可用于测量探针 - 靶杂交和检测的荧光团标记靶确定靶核酸序列的相对丰度。微阵列实验可以完成多个基因测试在并行，因为数组可能包含探头数以万计。一个简单的微阵列实验的布局示于图1。最近，它建立在我们和其他实验室，这些阵列是可重复使用的，这使得该技术相当高性价比即不同的RNA的分离和纯化技术已开发多年，包括C-TAB，SDS和GT方法4 - 8。此外，一些商业工具包也可提供。用于基因表达的高品质的RNA是非常重要的。因此，RNA分离方法被修改，以获得RNA的最大数量。同样，步骤进行cDNA制备cDNA的和标签被最小化。还通过内部内置软件包和工具进行9有效扫描后的数据正常化。 肺炎链球菌是一种革兰氏阳性的人类病原体是定植鼻咽和是多重感染，如肺炎，败血症，中耳炎的病因和脑膜炎10。该细菌可以利用各种所需的生长和存活11,12的营养素。大量的研究已经进行了对肺炎球菌氮代谢和调节强调氨基酸的重要性及其在毒力13,14作用。在这项研究中，S的转录反应肺炎改变L-丝氨酸，氨基酸大量存在的人血浆中的浓度，使用DNA微阵列的报道。 S的 ​​转录反应肺炎在L-丝氨酸（150μM）的最低浓度生长进行比较，在最大浓度（10毫米）的丝氨酸的生长。化学上确定的培养基（CDM或基本培养基）15被用于该研究，以控制丝氨酸的浓度。本研究的焦点是使该技术的用户友好的，并提供对数据归一化和分析不同的工具。因此，许多工具用于分​​析和数据解释被开发。 FIVA（函数信息查看器和分析器）提供了包含在集群有基因的信息处理平台相似的基因表达模式和构建功能简16。检察官是另一个软件包，有助于推测功能和基因17的注解标识。通过利用聚类方法公开提供了一种DNA结合位点的检测算法。基因的顺 -regulatory图案可以通过使用这种算法18进行投影。 Genome2D通过提供不同颜色范围来表征上的基因组中的地图19中的变化，基因表达水平提供了一个基于Windows的平台，可视化和转录组数据的分析。辣椒Web服务器提供，除了所有功能于一身的分析方法，一个工具箱开采调节子，推动者和转录因子结合位点20。间区域中的细菌基因组完整注释可以通过使用这个包来实现。生物学家可以大大辣椒中受益，因为它为他们提供了一个平台，设计性实验S以便虚拟信息可以在体外 20来确认。这些软件包显著有助于微阵列分析，其中大部分都是免费提供的，使数据标准化和分析非常可靠。

<table><tbody><tr><td>Acid phenol</td><td>SigmaAldrich</td><td>P4682</td><td></td></tr><tr><td>Roche RNA isolation kit</td><td>Roche Applied Science</td><td>11828665001</td><td></td></tr><tr><td>Glass beads</td><td></td><td>105015</td><td></td></tr><tr><td>Chloroform</td><td>Boom</td><td>92013505.1000.</td><td></td></tr><tr><td>IAA</td><td></td><td>106630</td><td></td></tr><tr><td>Nanodrop</td><td>Nanodrop</td><td>ND-100</td><td></td></tr><tr><td>Agilent BioAnalyser</td><td>Agilent</td><td>G2940CA</td><td></td></tr><tr><td>Superscript III</td><td>Life technologies Invitrogen</td><td>18080044</td><td></td></tr><tr><td>AA-dUTP</td><td>Life technologies Invitrogen</td><td>AM8439</td><td></td></tr><tr><td>DDT</td><td>Life technologies Invitrogen</td><td>18080044</td><td></td></tr><tr><td>First Strand buffer</td><td>Life technologies Invitrogen</td><td>18080044</td><td></td></tr><tr><td>NaOH</td><td>SigmaAldrich</td><td>S8045-1KG</td><td></td></tr><tr><td>HEPES</td><td>SigmaAldrich</td><td>H4034-500G</td><td></td></tr><tr><td>DyLight-550</td><td>Thermoscientiffic</td><td>62262</td><td></td></tr><tr><td>DyLight-650</td><td>Thermoscientiffic</td><td>62265</td><td></td></tr><tr><td>SHY Buffer</td><td>SigmaAldrich</td><td>H7033-125ML</td><td></td></tr><tr><td>Speedvac cooler</td><td>Eppendorf</td><td>RUGNE3140</td><td>Speedvac concentrator plus</td></tr><tr><td>Hybridization oven</td><td>Grant Boekel</td><td>Iso-20</td><td></td></tr><tr><td>Lifter-slips</td><td>Erie Scientific</td><td>25x60I-M-5439</td><td></td></tr><tr><td>Wipe</td><td>KIMTECH</td><td></td><td></td></tr><tr><td>SDS</td><td>SigmaAldrich</td><td>L3771-100g</td><td></td></tr><tr><td>SSC</td><td>SigmaAldrich</td><td>W302600-1KG-K</td><td></td></tr><tr><td>Genpix autoloader 4200A1</td><td>MSD analytical technologies</td><td></td><td>Microarray scanner</td></tr><tr><td>Sodium bicarbonate</td><td>SigmaAldrich</td><td>104766</td><td></td></tr></tbody></table>

a fast and reliable pipeline for bacterial transcriptome analysis case study: serine-dependent gene regulation in streptococcus pneumoniae

基因表达及其调控是了解细胞在不同条件下的行为非常重要。使用各种技术现今研究基因表达，但大部分是在提供全转录的表达的总体图象方面受到限制。 DNA微阵列提供了快速和经济研究的技术，这给全局基因表达的全面概述，并且具有的大量应用，包括鉴定新基因和转录因子结合位点，该细胞的转录活性的表征以及在分析数千个帮助基因（在单次实验）。在本研究中，从细胞收获到DNA微阵列分析细菌转录组分析的条件进行了优化。考虑到时间，成本和实验的精度，该技术平台被证明是非常有用的和普遍applicabale用于研究细菌transcriptoMES。在这里，我们通过对比S的转录反应进行DNA微阵列分析与肺炎链球菌作为一个案例研究肺炎生长在不同的L-丝氨酸的浓度在培养基中的存在。分离总RNA通过使用使用RNA分离试剂盒和RNA的质量Macaloid方法通过使用RNA质量检查试剂盒检查。制备cDNA用逆转录酶和该cDNA样品使用两个胺反应性荧光染料1标记。自制DNA微阵列载玻 ​​片用于标记的cDNA样品和微阵列数据的杂交通过使用cDNA微阵列数据前处理框架（Microprep）进行了分析。最后，网络-T是用来分析使用Microprep为统计学显著差异表达的基因的识别生成的数据。此外，在公司内部建立软件包（辣椒，FIVA，披露，检察官，Genome2D）被用来分析数据。

RNA，基因隔离和分析 L-丝氨酸是必需的氨基酸之一，其在人类血浆浓度从60-150微米的儿童和成人不相同。其中嘌呤和嘧啶的生物合成作用凸显其重要性在代谢和它的前体，以数个氨基酸（甘氨酸，半胱氨酸和色氨酸）。以研究L-丝氨酸对S的全转录的影响肺炎 D39野生型菌株，生长在CDM与针对该中的最大浓度（10毫米），在相同的培养基中生长的L-丝氨...

Watch this Scientific Journal Video about A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae at JoVE.com

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

This manuscript describes the use of state-of-the-art technology provided by DNA-microarrays. Microarrays provide an overview of the transcriptomic changes in bacteria incurred under a specific condition. Moreover, we highlight the ease by which large amounts of data can be analyzed by using convenient in-house developed software packages.

在丝氨酸依赖的基因调控:一个快速和可靠的管道细菌转录组分析案例研究<em&gt;肺炎链球菌</em

Gene expression and its regulation are very important to understand the behavior of cells under different conditions. Various techniques are used nowadays ...

a-fast-reliable-pipeline-for-bacterial-transcriptome-analysis-case

Research

JoVE Journal

Biology

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

10.3K 次观看.  University of Groningen. This manuscript describes the use of state-of-the-art technology provided by DNA-microarrays. Microarrays provide an overview of the transcriptomic changes in bacteria incurred under a specific condition. Moreover, we highlight the ease by which large amounts of data can be analyzed by using convenient in-house developed software packages. 

视频: A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

科研

遗传学

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

免疫与感染

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

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

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 ...

生物化学

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

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate&#160;as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 &#956;L) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 &#956;M) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput&#160;gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

用于合成生物学和天然产物应用的高产 链霉菌 转录翻译工具包

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a ...

生物工程

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

生物学

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

在血清型 1 型肺炎链球菌 菌株 519/43 中构建突变体

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of the disease.&#160;To study such processes, researchers can use high-throughput sequencing techniques (RNA-seq) that measure the dynamic changes of the host transcriptome at different stages of infection, clinical outcomes, or disease severity.This investigation can lead to a better understanding of the diseases, as well as uncovering potential drug targets and treatments. The protocol presented here describes a complete pipeline to analyze RNA-sequencing data from raw reads to functional analysis. The pipeline is divided into five steps: (1) quality control of the data; (2) mapping and annotation of genes; (3) statistical analysis to identify differentially expressed genes and co-expressed genes; (4) determination of the molecular degree of the perturbation of samples; and (5) functional analysis. Step 1 removes technical artifacts that may impact the quality of downstream analyses. In step 2, genes are mapped and annotated according to standard library protocols.&#160;The statistical analysis in step 3 identifies genes that are differentially expressed or co-expressed in infected samples, in comparison with non-infected ones. Sample variability and the presence of potential biological outliers are verified using the molecular degree of perturbation approach in step 4. Finally, the functional analysis in step 5 reveals the pathways associated with the disease phenotype. The presented pipeline aims to support researchers through the RNA-seq data analysis from host-pathogen interaction studies and drive future&#160;in vitro or in vivo experiments, that are essential to understand the molecular mechanism of infections.

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical approaches.

用于研究宿主-病原体相互作用的高通量转录组分析

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of ...

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is ...

在丝氨酸依赖的基因调控:一个快速和可靠的管道细菌转录组分析案例研究

摘要

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此视频中的章节

基于荧光的感染组织细菌基因调控方法

肺炎链球菌在鼻内殖民期间炎症反应的特征

MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合

细菌中的RIBO-seq:NGS测序样本收集和库准备协议

高通量设置的建立筛选小分子能调节C-二GMP信令

用于合成生物学和天然产物应用的高产链霉菌转录翻译工具包

全基因组基因缺失

在血清型 1 型肺炎链球菌菌株 519/43 中构建突变体

用于研究宿主-病原体相互作用的高通量转录组分析

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

在丝氨酸依赖的基因调控:一个快速和可靠的管道细菌转录组分析案例研究

摘要

探索更多视频

此视频中的章节

基于荧光的感染组织细菌基因调控方法

肺炎链球菌在鼻内殖民期间炎症反应的特征

MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合

细菌中的RIBO-seq:NGS测序样本收集和库准备协议

高通量设置的建立筛选小分子能调节C-二GMP信令

用于合成生物学和天然产物应用的高产 链霉菌 转录翻译工具包

全基因组基因缺失

在血清型 1 型肺炎链球菌 菌株 519/43 中构建突变体

用于研究宿主-病原体相互作用的高通量转录组分析

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

用于合成生物学和天然产物应用的高产链霉菌转录翻译工具包

在血清型 1 型肺炎链球菌菌株 519/43 中构建突变体