这项工作的经费由基金会贝当古舒莱尔支持贝当古巴黎队iGEM大赛提供。我们感谢INSERM U1001科研单位，尚塔尔对于LOTTON技术援助和咨询。噬菌体Litmus28i_J23115-B0032-GFP是由莫妮卡·奥尔蒂斯和斯坦福大学的德鲁恩迪提供。

1.设计轴承斯尔纳沉默盒噬菌粒载体的构建<ol><li>斯尔纳沉默盒8的从头设计<ol><li>识别mRNA的完整序列使用DNA序列数据库被沉默。以产生靶序列，请注意编码序列的前24个碱基对，从位置+1至+24与起始密码子（ 如，ATG）开始。 注意:沉默效率较低时的其他网站或mRNA的片段为目标8。 </li><li>取靶序列的反向互补，以产生对的sRNA盒导向序列。请参阅表1为氯霉素乙酰转移酶（CAT）目标和指南序列的例子。 </li><li>设计292 bp的完整的sRNA表达盒，安排PR启动子，指导序列，HFQ蛋白结合串联域和T1 / TE转录终止序列（ 表2）。 </li><li>添加所选择的附加的克隆位点，以方便的sRNA盒克隆到目标矢量。 </li><li>通过商业基因合成或类似的方法来获得完整的斯尔纳磁带，它克隆与功能F1复制起点11的噬菌体载体。看到的最终噬菌粒载体的完整序列支持信息。 </li></ol></li><li>改变使用基于PCR的定点诱变12现有斯尔纳表达盒靶向序列<ol><li>识别在现有的sRNA表达盒24 bp的指南序列。注:注释pAB.001质粒，在这项工作中使用，可作为补充的序列文件。 </li><li>设计正向和反向与现有的sRNA盒侧翼新的24 bp的指南序列同源的短区域的引物。获得通过商业寡核苷酸合成引物。 注意:定点诱变引物设计是depic泰德于图2在表3中提供精确的引物模板的序列。 </li><li> E.准备的5毫升培养大肠杆菌携带模板斯尔纳表达噬菌粒。生长细胞过夜，在37℃振荡，在LB培养基中以适当的抗生素。 </li><li>提取和使用DNA小量试剂盒或类似的方法12中的5毫升细菌培养净化模板斯尔纳表达噬菌粒。 </li><li>准备使用的模板的sRNA表达噬菌粒和高保真聚合酶，一种具有前向和一个与反向引物（ 表4）两个PCR反应。使用的PCR条件的建议通过聚合酶供应商（ 表5）。增加模板浓度比标准反应10-50x更高占的事实，单引物反应不产生指数扩增。 </li><li>结合上述两个PCR反应在一个离心管中。 Annea微升的产品通过加热到98℃的沸水浴中。放置微量离心管在水浴后，立即取出热源并允许浴超过1-2小时缓慢回到室温。 </li><li>为了消除未突变模板斯尔纳表达噬粒，加入1微升。的DpnI限制性内切酶的混合物中，并孵育在37℃下1小时，或由制造商为完全消化所建议的时间。 注意:的DpnI消化只有甲基化靶位点，这是存在于主机的复制噬菌粒，但不PCR产物。 </li><li>变换购买或准备13化学感受态大肠杆菌 1-5微升退火的PCR产物。通过在含有适当的抗生素的LB琼脂平板选择性电镀隔离转化菌株的单菌落。 </li><li>以验证正确的指导序列的掺入，筛选通过菌落PCR所得菌落。用200μl的枪头，合作从单个转化的菌落llect细胞的少量。马克和保留原来的殖民地核实后下游使用。 </li><li>收集的细胞添加至50μl的无核酸酶水在一个离心管中。吹打向上和向下混合。 </li><li>使用台式热循环或沸水浴，通过加热溶解细胞到95℃2分钟。 </li><li> PCR的扩增使用1μl的热裂解的细胞作为DNA模板噬菌粒区。在表6和7中提供的PCR条件和热循环协议。请参阅验证引物序列补充pAB.001序列文件。 </li><li>测序的PCR产物以验证正确的指导序列的掺入。 </li><li>接种大肠杆菌的5毫升培养大肠杆菌克隆携带序列验证斯尔纳表达噬菌粒。生长细胞过夜，在37℃振荡，在选择性LB培养基。 </li><li>准备甘油股票的序列验证克隆。添加750微升过夜培养物，以250μl的60％的甘油在一螺丝帽冷冻管中。 </li><li>在-80℃保存甘油下去。在过夜培养的剩余部分可被用作在步骤2中的sRNA表达噬菌粒的来源。 </li></ol></li></ol> M13包装库存噬菌粒2.生产和收获<ol><li> E.准备的5毫升培养大肠杆菌携带斯尔纳表达噬菌粒。生长细胞过夜，在37℃振荡，在LB培养基中以适当的抗生素。注意:的sRNA表达噬菌粒可通过从头克隆如步骤1.1.5描述从现有噬菌粒来获得，或修改，并在步骤1.2.16收获。 </li><li>同样准备E的5毫升培养大肠杆菌携带M13KO7辅助质粒。生长细胞过夜，在37℃振荡，在选择性LB培养基。 </li><li>提取和纯化的斯尔纳expressioÑ ​​噬菌粒和用DNA提取试剂盒或类似的方法12辅助质粒。 </li><li> Cotransform购买或制备13化学感受态大肠杆菌与每个的sRNA表达噬菌粒和辅助质粒的1微升。通过这两个结构选择性抗生素的LB琼脂电镀选择cotransformants。 </li><li>从在LB的共转化株与选择性抗生素的单菌落制备10ml的培养。在37℃孵育摇8-12小时或过夜。 </li><li>离心培养3300 XG 10分钟。收集通过0.2微米的过滤器将上清液过滤。注意:在媒体泄漏的情况下，请用稀释的漂白剂（0.5％），该地区摧毁感染性噬菌体颗粒。 </li><li>储存在4℃下将包装噬菌粒滤液。注:样品可以维持数天至数周而不丧失活性。 </li></ol> 3. F +靶细胞的制备沉默<ol><li>确定是否要针对沉默细胞表达歼14菌毛。如果F菌毛已经存在，继续步骤4。 注:E.常见的实验室菌株大肠杆菌被注解为F +或F&#39;以指示在其基因组上或质粒在F菌毛的存在。 </li><li> E.获得的F +菌株大肠杆菌如TOP10F&#39;。 注意:确保目标菌株携带一个独特的抗性标记，以从结合后的F-质粒供体分离。 </li><li>要通过与F +菌株结合引进˚F菌毛，准备5毫升培养目标应变和F-质粒捐助14的都有。生长细胞过夜，在37℃振荡，在LB培养基中以适当的抗生素。 </li><li>第二天，稀释两种菌株1:100在5ml选择性的LB，并继续培养，在37℃振荡。 </li><li>通过测量选择确定细胞的生长相在使用台式分光光度计600纳米（OD 600）文化的iCal密度。培养得到的细胞约2小时，直到0.3的OD 600，指示数期生长15。 </li><li>在微量离心管准备3偶联反应:0.5毫升F-质粒供体+0.5毫升靶菌株加入0.5ml的F-质粒供体+ 0.5毫升LB培养基培养基（阴性对照）和0.5ml靶应变+ 0.5毫升LB培养基培养基（阴性对照）。允许缀合，在37℃振荡进行2小时。 </li><li>板100微升在选择性LB琼脂每个缀合反应与具体到F质粒的抗生素（通常四环素）和目标菌株。板阴性对照反应，以确认没有捐赠者或受体菌表达两种抗生素的抗性。 </li></ol> 4.感染了沉默噬菌粒包装<ol><li>接种F +靶细胞的单个菌落到LB中介的5毫升培养一个适当的抗生素。在37℃振荡孵育过夜。 </li><li>第二天，稀释F +靶细胞1:100在5ml选择性LB ​​培养基，并继续培养，在37℃振荡。 </li><li>通过使用台式分光光度计测定培养物的光密度在600nm（OD 600）测定细胞的生长阶段。培养得到的细胞约2小时，直到0.3的OD 600，指示数期生长15。注:在F菌毛和感染效率表达最高的是日志阶段。 </li><li>添加M13-包装噬​​菌粒（来自步骤2.6）与靶细胞以1:1的体积比:100达到目标人口的近99％的感染。允许感染在37℃振荡30-60分钟进行。 </li><li>根据所选择的方法测定了的sRNA沉默表型。 注意:对于荧光蛋白靶标，消音效果可以定量直接荧光10。交替，表型分析可用于观察基因敲除8的表型的影响。 </li><li>准备甘油股票以下步骤1.2.14-1.2.16的斯尔纳表达噬菌粒主机。注意:噬菌粒将在宿主菌株无限期传播，并且可以与抗生素类似于传统的质粒被维持。 </li></ol>

<ol>
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The authors have nothing to disclose.

相比于不相关的对照本方法实现了mKate荧光水平减少80％。这是符合其他RNA敲除的方法，其中完整的沉默不遵守和50-90％的效率是典型的16,17。在表型水平，CAT定位击倒能够显著衰减氯霉素抗性，且消除它在某些条件下。 只有几个小时后感染（ 图3B）后，击倒表型是在群体水平检测。这说明了基于噬菌体的输送的一个重要特征:高的击倒频率可以直接在分批培养无需事先遗传修饰而获得。不像使用质粒转化或基因组整合常规遗传修饰，噬菌体感染不要求一个人口从单个孤立菌落再生长。这使得噬菌体感染的效果是EXPLORED与复杂的空间动态11的人群，像生物膜18，或者在基因混合自然种群19预先存在的空间结构。 该方法的关键步骤是在高滴度的生产包装噬菌粒。与噬菌体颗粒的生产相关的代谢负担可能会导致在噬菌粒生产菌株的突变或质粒损失率高。建议在噬菌粒生产菌株从单个共转化菌落直接培养，不冷藏之前，噬菌体收获冷冻或子培养。引入噬菌粒和辅助质粒到大肠杆菌时，也可以观察到共转化的效率低同时大肠杆菌 。在这种情况下，更高的效率可以通过首先将所述辅助质粒，然后制备感受态细胞的轴承用于与噬菌粒随后转化辅助质粒而获得。 帕gemid感染或的sRNA表达还对靶细胞的可检测的代谢负担，并且可能导致一些表型扰动。例如，在mKate2荧光的减少观察到即使当细胞感染噬菌粒靶向CAT（ 图3）。与M13感染是没有想到来触发E.全身应激反应大肠杆菌 20，但也可以间接地改变转录模式。另外，包括在噬菌粒的GFP或氨苄青霉素抗性标记可能会争夺资源的细胞，减少mKate2表达和9。最后，的sRNA盒本身可以全局通过滴定HFQ蛋白改变基因表达谱，或通过脱靶mRNA的沉默。脱靶效应在体内 RNA干扰21-23常见，但他们还没有被系统地研究了这个系统。 这种方法的一个限制是该感染艾菲效率低于100％，允许一些未感染的细菌中的人口持续。这项工作，并早期工作10的结果表明，未感染细胞代表最终人口的1-10％，并且负责大多数观察到的nonsilenced表型。各种路由M13-电阻是已知的，与菌毛表达24的最常见的突变损失。在这些限制的光，控制应该被用来确认高感染率和敲低效率。 在某些应用中另一个潜在的限制是污染辅助噬菌体偶尔传输。虽然M13K07包含突变的包装信号，它可以被包装成在低频噬菌体衣壳，并转移到受感染的人群中，导致胜任噬菌体生产和噬菌体超出了最初的感染事件25继续扩散的细胞。修改辅助噬菌体已被证明有效在以降低生产噬菌体26的成本降低了非特异性的包装，虽然有时。 改造噬菌体已成为大肠杆菌的必备工具大肠杆菌合成生物学，允许快速交货的新基因，以不断增长的人口。最近的工作已经产生细胞间通信电路11或表达的转录因子来抑制抗生素耐药性的途径27。这里介绍的协议增加了工具，允许通过编程的RNA细菌生理控制不断增长的集合。 CRISPR-CAS核酸酶，突变以消除核酸酶活性时，已经示出在RNA的引导基因靶17,28到抑制转录。与此相反，的sRNA沉默工作在翻译水平，并且不需要外源蛋白的表达。下一代生物技术可联合转录和翻译控制与噬菌体介导的递送到复杂苯氧编程类型的实时性。

的RNA介导的基因击倒进行两个阶段。首先，将RNA分子引入到细胞系或研究的有机体。第二，内源性RNA结合蛋白促进RNA的目标识别和产生的消声效果。所有的RNA拦截技术受益于合成sRNAs的定制性质，可以容易地制造，以匹配感兴趣的特定目标。然而，RNA摄取和沉默的分子细节横跨模型系统广泛地变化，限制在哪里以及如何的RNA击倒可以应用。 在线虫，双链RNA（dsRNA）的分子可直接在介质或通过用双链RNA表达大肠杆菌的群体喂食蠕虫引入大肠杆菌细胞1,2-，在果蝇中 ，RNA干扰，可以通过用双链RNA 3显微注射的胚胎通过简单地添加的dsRNA至培养基4来实现，或在细胞系中实施。在哺乳动物细胞系，合成小干扰RNA（siRNA）可以通过电穿孔1,2,5递送到活细胞，封装在脂质体3,6，或从DNA质粒载体4,7-表达。一旦RNA种类到达胞质溶胶，RNAi途径依赖于RISC复合物处理的ds RNA，促进靶的反义识别，并催化翻译抑制，mRNA降解，或异染色质形成，这取决于主机上。 由于这些要求的，古典的RNAi只能在有效地吸收外源RNA并表达RISC或类RISC活性有机体进行。值得注意的是，这不包括模型细菌E.大肠杆菌 ，它缺乏RNAi途径。然而，最近在合成生物学的发展提供必要的工具来解决这两个问题，交付和沉默问题。 在这个协议中，斯尔纳构建在大肠杆菌表达来自大肠杆菌的DNA载体交付给李咏使用M13噬菌体/辅助系统细胞。噬菌粒是复制的噬菌体衍生的F1起源的质粒。辅助质粒，在此情况下M13KO，进行所有以产生病毒颗粒所要求的机械，但本身不是胜任复制和包装。当噬菌粒和辅助质粒被共转化，单独噬菌粒是在F1原点复制，包装和分泌的。该矢量噬菌体是那么能干感染现场E.通过F菌毛的大肠杆菌 。 在这个系统中，沉默效应是由定制的sRNA盒结合的靶结合序列的支架序列产生。靶结合序列是反义的mRNA靶24个碱基对，通常是在核糖体结合位点（RBS）。该支架序列，由娜和他的同事开发出8，包含MICC，小调控RNA内源性大肠杆菌提取的HFQ结合基序大肠杆菌 。该HFQ蛋白刺激RNA的RNA结合蛋白g且mRNA降解，供应在该系统类似于RISC中的RNAi作用。 图1描绘了用于噬菌体介导的sRNA击倒，包括的sRNA盒结构，噬菌粒矢量，和消音机构的完整方案。 作为一种方法来调节在大肠杆菌中基因表达的大肠杆菌 ，斯尔纳沉默是简单，快速和灵活。有针对性的E.大肠杆菌是不是负担超出传播噬菌粒表达斯尔纳。这可能是在合成生物学或基础研究，其中较大的异源构建体的表达可以应变蜂窝资源9的上下文相关的。与新的目标噬菌粒可以用单个的PCR来制备和噬菌粒转化后收获一天。最后，几乎所有的表达可以有针对性。所述的sRNA调节盒（在标准的质粒）已经显示出对各种目标在代谢与典型的压制水平&gt; 90％8工作。 10先前的工作扩展。首先，一个包装噬菌粒引入到大肠杆菌的分批培养大肠杆菌细胞并用于沉默荧光蛋白mKate2的表达。后续的荧光变化进行实时监测。第二，撞倒CAT基因中示出减少在琼脂平板上的表型氯霉素抗性。在这两种情况下，噬菌粒本身携带GFP标记，允许待测量独立的击倒效率感染率。

<table><tbody><tr><td>Plasmid Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>DpnI Enzyme</td><td>NEB</td><td>R0176S</td><td></td></tr><tr><td>Phusion High Fidelity Polymerase</td><td>NEB</td><td>M0530S</td><td></td></tr><tr><td>Taq 2x Master Mix</td><td>NEB</td><td>M0270L</td><td></td></tr><tr><td>M13KO7 Helper Phage</td><td>NEB</td><td>N0315S</td><td></td></tr><tr><td>DH5&amp;alpha; Competent Cells</td><td>Life Technologies</td><td>18265-017</td><td></td></tr><tr><td>TOP10F&#039; Cells</td><td>Life Technologies</td><td>C3030-03</td><td></td></tr><tr><td>LB Broth</td><td>Sigma</td><td>L3022-250G</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9393-5G</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma</td><td>60615-5G</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378-5G</td><td></td></tr><tr><td>Tetracycline</td><td>Sigma</td><td>87128-25G</td><td></td></tr></tbody></table>

phage-mediated delivery of targeted srna constructs to knock down gene expression in e. coli

的RNA介导的击倒被广泛用于控制基因表达。该技术多功能系列采用可与任何序列合成和设计，以配合针对任何沉默基因的短RNA（斯尔纳）的。因为的sRNA构建可以引入到许多细胞类型直接或使用各种载体，基因表达可以在活细胞中，而不费力遗传修饰被抑制。最常见的RNA拦截技术，RNA干扰（RNAi），利用了靶mRNA的内源RNA诱导的沉默复合物（RISC）介导的序列识别和切割的。因此这种技术的应用只限于RISC的表达生物体，主要是真核生物。近日，新一代生物技术RNA已经开发的替代机制，通过RNA调控基因表达，在细菌，因此成为可能RNA介导的基因击倒。在这里，我们介绍了一种沉默基因EXPRES锡永在E.大肠杆菌的功能类似于RNA干扰。在这个系统中的合成噬菌粒被设计来表达的sRNA，其可设计为靶向任何序列。表达构建体递送到大肠杆菌的群体的大肠杆菌细胞与非裂解M13噬菌体，在这之后能够稳定地复制作为质粒。反义识别与靶mRNA的沉默由HFQ蛋白质，内源性大肠杆菌介大肠杆菌 。该协议包括用于设计反义的sRNA，构建噬菌粒载体，包装噬菌粒到M13噬菌体，感染制备活细胞群体，并执行感染本身的方法。荧光蛋白mKate2和抗生素抗性基因氯霉素乙酰转移酶（CAT）定位到产生代表数据和量化击倒效力。

在液体介质mKate2荧光沉默 图1描绘了用于在这项工作中所描述的sRNA介导的击倒，包括的sRNA盒设计，噬菌粒矢量，和沉默机制的方案。以下协议1.2，质粒pAB.001的斯尔纳沉默盒改变目标mKate。该斯尔纳盒原料合成并克隆到噬菌粒Litmus28i_J23115-B0032-GFP，莫妮卡奥尔蒂斯和德鲁恩迪11的礼物。此噬菌粒携带GFP表达和卡那霉素抗性标记，允许被跟踪成功感染。制备包装噬菌股以下协议2。 E.的衍生物大肠杆菌 K12 MG1655背着一个组成型表达，染色体整合mKate2标记是由共轭的噬菌体感染准备<html>用以下方案3细胞生长至对数中期，介绍以下协议4.噬菌粒感染后噬菌粒一个F质粒供体菌株，200微升的培养物转移到荧光板读数器和荧光24小时连续监测。 图3示出的sRNA介导的沉默对mKate2表达的影响。感染与抗mKate2噬菌粒的菌株没有表现出可检测的mKate2荧光超过本底。相比之下，这株没有表达GFP标记，表明噬菌体的成功吸收。未感染的对照细胞产生mKate2荧光但不GFP。附加控制，其中，反mKate2靶向域与一个序列靶向的CAT取代，对mKate2荧光没有影响。 氯霉素抗性的琼脂平板上沉默内容"FO:保together.within页="1"&gt;以下协议1.1，制作一个斯尔纳沉默卡带目标CAT 大肠杆菌 K12 MG1655背着一个组成型表达，染色体整合CAT标志是为准备的衍生物。噬菌体感染通过与以下方案3.将细胞在F-质粒供体菌株偶联生长到对数中期和噬菌粒在37℃下引入以下协议4.温育1小时后，感染的细胞进行系列稀释，并在镀的氯霉素浓度。将板范围温育过夜，并抗性细胞中的每一个氯霉素浓度的比例通过计数菌落形成单位（CFU的）次日确定。 图4示出的sRNA介导的沉默对氯霉素抗性的表型的影响。未感染的细胞，或感染噬菌粒靶向mKate2细胞，分别以抗氯霉素在所有测试浓度。相比之下，感染了针对噬菌粒细胞CAT在低浓度氯霉素减少显示出生存，近99％的较高浓度杀。添加氨苄青霉素，要选择只携带细菌噬菌体，减少氯霉素生存检测不到的水平。这与早期的工作一致表示从噬菌体感染的逃避逃跑沉默10共同路线。 <img alt="图1" src="/files/ftp_upload/53618/53618fig1.jpg" /> 图1:在 大肠杆菌 的基因沉默 大肠杆菌 用的sRNA表达盒由M13噬菌体递送的的sRNA盒是由4个模块:所述PR启动子（来自λ噬菌体来源的组成型启动子），一个24bp的靶向结构域，HFQ结合域从MICC提取和转录终止子8 </SUP&gt;。磁带盒被掺入噬菌粒轴承M13 F1复制起点和包装到使用辅助噬菌体M13KO7的M13衣壳。包装噬菌粒能够感染大肠杆菌大肠杆菌表达在F菌毛，其中斯尔纳表达开始。该斯尔纳然后招募HFQ蛋白（红色表示），并结合反义靶mRNA核糖体结合位点附近，导致翻译抑制和mRNA降解。 <a href="https://www.jove.com/files/ftp_upload/53618/53618fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53618/53618fig2.jpg" /> 图2:引物的设计和的sRNA目标站点的定点诱变的引物被设计具有部分同源性的现有的sRNA盒。正向引物含有20bp的同源的5&#39;端的PR启动子，接着表示新的指南序列24碱基，然后18碱基对同源的3&#39;末端的HFQ结合结构域。反向引物是正向引物的确切反向互补，与现有的sRNA盒侧翼的新指南序列的反向互补同源性的区域。确切的引物序列在表3中给出。与正向和反向引物的独立的单引物的PCR反应产生具有所需修饰的序列的直链，单链DNA。退火正向和反向的反应产物，按照如在协议中所述的清理，结果在双链DNA质粒带有所需修改斯尔纳磁带。 <a href="https://www.jove.com/files/ftp_upload/53618/53618fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53618/53618fig3.jpg" /> 图3:K一个染色体整合mKate2荧光记者nockdown。E.大肠杆菌 MG1655 K12表达mKate2要么感染与抗mKate2噬菌粒（绿线和棒）左未处理（红色线和条），或感染了控制噬菌粒靶向CAT（蓝线和条）。 （ 一 ）未经处理的E.大肠杆菌上升到更高的饱和密度，说明代谢成本噬菌体感染。虚线表示的3次重复的标准偏差。 （B）中的mKate2信号降低到接近背景水平在抗mKate2处理应变，而不是在对照菌株。 （C）GFP荧光，也由噬菌粒携带，只在噬菌粒处理的对照是可检测的。 （D，E）最终荧光读数生长24小时后标准化为外径。 GFP信号，指示噬菌体感染时，在未经处理的控制缺席，而且还大幅减少以下抗猫phagemid治疗。这可以指示噬菌粒的脱靶效应。所述mKate2信号减少了抗mKate2噬菌粒治疗相比未处理的对照。在CAT-针对性的控制噬菌显示对mKate2荧光没有影响。误差棒代表3次重复的标准差。 <a href="https://www.jove.com/files/ftp_upload/53618/53618fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53618/53618fig4.jpg" /> 图4:CAT的击倒恢复氯霉素敏感性遗传抗性种群E.。 大肠杆菌 MG1655 K12表达染色体整合CAT基因不进行处理（红色条），用控制噬菌粒靶向mKate2（绿色柱）处理，或用表达抗CAT的sRNA（蓝色条）噬菌粒处理。 1小时后感染，生存能力上表示蚂蚁ibiotics被连续稀释和电镀评估。用抗CAT噬菌粒处理过的菌株通过在较高浓度氯霉素显著杀死（&gt; 90％），而对照处理不受影响。加入氨苄青霉素到培养板正选择用于噬菌粒感染和消除未感染的细胞。在这些条件下，抗CAT处理后没有观察到氯霉素抗性菌落。这表明大多数幸存者代表感染的故障，而不是沉默的故障。误差棒代表3次重复的标准差。 <a href="https://www.jove.com/files/ftp_upload/53618/53618fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> CAT靶序列</td><td> 5&#39; - ATGGAGAAAAAAATCACTGGATAT - 3&#39; </td></tr><tr><td> CAT指导序列</td><td> 5&#39; - ATATCCAGTGATTTTTTTCTCCAT - 3&#39; </td></tr></tbody></table> 表1:示例TARGET和指导序列的CAT基因注意的反向互补关系。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> PR启动</td><td colspan="5"> TAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC </td></tr><tr><td>指导序列</td><td colspan="5"> ATATCCAGTGATTTTTTTCTCCAT </td></tr><tr><td> HFQ结合域</td><td colspan="5"> TTTCTGTTGGGCCATTGCATTGCCACTGATTTTCCAACATATAAAAAGACAAGCCCGAACAGTCGTCCGGGCTTTTTT TCTCGAG </td></tr><tr><td> T1 / TE终结者</td><td colspan="5"> CTCGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTTTTGTCGGTGAA CGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA </td></tr></tbody></table>表2:所述的sRNA盒式序列组件每个序列写入5&#39;-3&#39;。完整的磁带是这4个元素的串联，以便与包括BP 292。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>正向引物</td><td> 5&#39; - CTGGCGGTGATAATGGTTGC [GUIDE] TTTCTGTTGGGCCATTGC - 3&#39; </td></tr><tr><td>反向引物</td><td> 5&#39; - GCAATGGCCCAACAGAAA [TARGET] GCAACCATTATCACCGCCAG - 3&#39; </td></tr></tbody></table> 表3:引物的设计改变现有引导元件的正向引物包括在过去的20碱基对的PR启动子，新的指导序列，和HFQ结合结构域的前18个碱基对。靶序列是指导序列的确切反向互补。反向引物是正向引物的确切反向互补。 </p&gt; <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>零件</td><td>卷</td></tr><tr><td>模板质粒</td><td> 500纳克</td></tr><tr><td> 10μM底漆</td><td> 2.5微升</td></tr><tr><td> 10毫米的dNTP </td><td> 0.4微升</td></tr><tr><td>高保真聚合酶（2U /微升） </td><td> 0.5微升</td></tr><tr><td> 5X PCR缓冲</td><td> 10微升</td></tr><tr><td>总成交量</td><td> 50微升</td></tr></tbody></table> 表4:推荐条件的单引物诱变的PCR。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>步</td><td>温度</td><td>时间</td></tr><tr><td>初始变性</td><td> 98℃ </td><td> 30秒</td></tr><tr><td rowspan="3"> 30个循环</td><td> 98℃ </td><td> 10秒</td></tr><tr><td> 55°C </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 120秒</td></tr><tr><td>最终延伸</td><td> 72℃ </td><td> 300秒</td></tr><tr><td>存储</td><td> 10℃ </td><td></td></tr></tbody></table> 表5:推荐热循环协议的单引物诱变的PCR。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>零件</td><td>卷</td></tr><tr><td>模板DNA </td><td> 1微升</td></tr><tr><td> 10μM正向引物</td><td> 0.5微升</td></tr><tr><td> 10μM反向引物</td><td> 0.5微升</td></tr><tr><td> Taq酶2X预混</td><td> 25微升</td></tr><tr><td>无核酸酶的水:- [R </td><td> 23微升</td></tr><tr><td>总成交量</td><td> 50微升</td></tr></tbody></table> 表6:建议的条件为序列验证PCR。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>步</td><td>温度</td><td>时间</td></tr><tr><td>初始变性</td><td> 95℃ </td><td> 30秒</td></tr><tr><td rowspan="3"> 30个循环</td><td> 95℃ </td><td> 30秒</td></tr><tr><td> 55°C </td><td> 30秒</td></tr><tr><td> 68℃ </td><td> 30秒</td></tr><tr><td>最终延伸</td><td> 68℃ </td><td> 300秒</td></tr><tr><td>存储</td><td> 10℃ </td><td></td></tr></tbody></table> 表7:建议热循环仪专业版母育的序列验证PCR。

Watch this Scientific Journal Video about Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli at JoVE.com

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达<em&gt;电子。大肠杆菌</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

phage-mediated-delivery-targeted-srna-constructs-to-knock-down-gene

Research

JoVE Journal

Biology

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

12.5K 次观看.  Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes. We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

视频: Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

高效率，站点特定的贴壁细胞转染siRNA的微电极阵列（MEA）

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

科研

生物工程

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

遗传学

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by pathogenic and virulent bacteria of the genus Leptospira, either by direct contact with the bacteria or indirectly by exposure to contaminated water or soil. Domestic and wild animals act as reservoir hosts of infection, shedding leptospires from colonized renal tubules of the kidney, via urine, into the environment. The generation of mutant strains of Leptospira is critical to evaluate and understand pathogenic mechanisms of infection. CRISPR interference (CRISPRi) has proven to be a straightforward, affordable, and specific tool for gene silencing in pathogenic Leptospira. Therefore, the methodological details of obtaining the plasmid constructs containing both dCas9 and guide RNA, delivery of plasmids to Leptospira by conjugation with the E. coli strain &#946;2163, and transconjugant recovery and evaluation, will be described. In addition, the recently described Hornsby-Alt-Nally (HAN) media allows for the relatively rapid isolation and selection of mutant colonies on agar plates.

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira

Here, the application of CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Leptospira cells are transformed by conjugation with plasmids expressing dCas9 (catalytically "dead" Cas9) and a single-guide RNA (sgRNA), responsible for base pairing to the desired genomic target. Methods to validate gene silencing are presented.

CRISPR干扰（CRISPRI）在瘦素病原物种中基因沉默的应用

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

生物学

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

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 ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1 
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
Transduction is a form of genetic exchange between bacteria that utilizes bacteriophages, or phages, a class of virus that infects exclusively prokaryotic organisms. This form of DNA transfer, from one bacterium to another by way of a phage, was discovered in 1951 by Norton Zinder and Joshua Ledererg, who termed the process &#34;transduction&#34; (1). Bacteriophages were first discovered in 1915 by British bacteriologist Frederick Twort, then independently discovered again in 1917 by French-Canadian microbiologist Felix d&#39;Herelle (2). Since then, the structure and function of these phages have been widely characterized (3), dividing these phages into two classes. The first of these classes are the lytic phages which upon infection multiply within the host bacterium, disrupting the bacterial metabolism, lysing the cell, and releasing progeny phage (4). As a result of this anti-bacterial activity and the increasing prevalence of antibiotic-resistant bacteria, these lytic phages have recently proved useful as a substitute treatment for antibiotics. The second of these classes are the lysogenic phages which can either multiply within the host via the lytic cycle or enter a quiescent state in which their genome is integrated into that of the host (Figure 1), a process known as lysogeny, with the ability for phage production to be induced in multiple later generations (4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10517/10517fig1.jpg" /> 
Figure 1: Infection of host cell by bacteriophage. Adsorption by the phage to the bacterial cell wall via interactions between the tail fibers and receptor (purple). Once on the cell surface, the phage is irreversibly attached to the bacterial cell using the base plate (black) which is moved to the cell wall by the contractile sheath (yellow). Phage genome (red) then enters the cell and integrates into the host cell genome.
While bacterial transduction is a naturally occurring process, using modern technology it has been manipulated for the transfer of genes into bacteria in the laboratory setting. By inserting genes of interest into the genome of a lysogenic phage, such as phage, one is able to transfer these genes into the genomes of bacteria and consequently express them within these cells. While other methods of gene transfer, such as transformation, use a plasmid for gene transfer and expression, the insertion of the phage genome into that of the recipient bacterium not only has the potential to confer new traits to this bacterium, but also allows for naturally occurring mutations and other factors of the cellular environment to alter the function of the transferred gene.
Compared to other methods of horizontal gene transfer, such as conjugation, transduction is fairly flexible in the criteria required for donor and recipient cells. Any genetic element that can fit inside the genome of the phage being used can be transferred from any strain of donor bacteria to any strain of recipient bacteria as long as both are permissive to the phage, requiring the expression of necessary phage receptors on the cell surfaces. Once this gene is moved out of the donor genome and packaged into the phage, it can be transferred to the recipient. Following transduction, it is necessary to select for recipient bacteria that contain the gene of interest have to be selected for. This could be done by use of a genetic marker, such as a FLAG-tag or polyhistidine-tag, to mark the gene of interest, or the intrinsic function of the gene, in the case of genes that encode for antibiotic resistance. Additionally, PCR could be used to further confirm successful transduction. By using primers for a region within the gene of interest and comparing signal to a positive control, bacteria that has the gene of interest, and a negative control, bacteria that underwent the same steps as the transduction reaction with no phage. While bacterial transduction is a useful tool in molecular biology, it has and continues to play an important role in the evolution of bacteria, particularly with regard to the recent rise of antibiotic resistance.
In this experiment, bacterial transduction was used to transfer the gene encoding for resistance to the antibiotic ampicillin from the W3110 strain of E.coli to the J53 strain via the P1 bacteriophage (5). This experiment consisted of two main steps. First, the preparation of P1 phage containing the ampicillin resistance gene from the donor strain. Second, the transfer of this gene to the recipient strain by transduction with the P1 phage (Figure 1). Once carried out, the successful transfer of the ampicillin resistance gene could be determined by qPCR (Figure 2). If transduction was successful, the J53 strain of E. coli would be resistant to ampicillin, and the gene conferring this resistance detectable by qPCR. If unsuccessful, there would be no detection of the ampicillin resistance gene and ampicillin would still function as an effective antibiotic against the J53 strain.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10517/10517fig2.jpg" /> 
Figure 2: The confirmation of successful transduction by qPCR. By comparing the Cq values detected for the gene of interest from the transduction reaction and the negative control reaction, and normalizing these values against a housekeeping gene, one was able to confirm that bacterial transduction was successful.

Phage Transduction: Transferring Ampicillin Resistance in E. coli

噬菌体转导:将氨基青霉素耐药性从捐赠者转移到受体大肠杆菌的方法

Source: Alexander S. Gold1, Tonya M. Colpitts1
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases ...

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RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli

Source: Kolundzic, E., et al. <a href="https://www.jove.com/video/56889/application-rnai-heat-shock-induced-transcription-factor-expression">Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in&#160;C. elegans</a>.&#160;J. Vis. Exp.&#160;(2018).
This video describes the principles behind RNAi treatment in C. elegans and demonstrates a protocol to knockdown lin-35 in a transgenic worm strain.

用于秀丽隐杆线虫饲喂的 RNAi 铺板:一种在大肠杆菌中诱导靶标 dsRNA 表达的技术

Source: Kolundzic, E., et al. Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in&#160;C. ...

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达

摘要

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此视频中的章节

高效率，站点特定的贴壁细胞转染siRNA的微电极阵列（MEA）

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

CRISPR干扰（CRISPRI）在瘦素病原物种中基因沉默的应用

细菌提供的RNAi效应：Transkingdom RNA干扰

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

全基因组基因缺失

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药

噬菌体转导:将氨基青霉素耐药性从捐赠者转移到受体大肠杆菌的方法

用于秀丽隐杆线虫饲喂的 RNAi 铺板:一种在大肠杆菌中诱导靶标 dsRNA 表达的技术

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达

摘要

探索更多视频

此视频中的章节

高效率，站点特定的贴壁细胞转染siRNA的微电极阵列（MEA）

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

CRISPR干扰（CRISPRI）在瘦素病原物种中基因沉默的应用

细菌提供的RNAi效应：Transkingdom RNA干扰

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

全基因组基因缺失

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

噬菌体转导:将氨基青霉素耐药性从捐赠者转移到受体大肠杆菌的方法

用于秀丽隐杆线虫饲喂的 RNAi 铺板:一种在大肠杆菌中诱导靶标 dsRNA 表达的技术

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药