Name: inducing a site specific replication blockage in e. coli using a fluorescent repressor operator system
Uploaded: 2016-08-21T15:00:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System at JoVE.com

This work was supported by the Australian Research Council [DP11010246].

1.阻止复制与FROS <ol><li>诱导复制遇阻<ol><li>稀释大肠的新鲜过夜培养（为0.17μmKH 2 PO 4，0.72 MK 2 HPO 4 0.1％胰蛋白胨，0.05％酵母提取物，0.1％氯化钠，）用抗生素如需要大肠杆菌菌株携带TETO阵列和PKM1 18至OD 600nm处 = 0.01在稀复合培养基供选择。 注意:不要以供选择添加四环素，因为它不与本系统相兼容。使培养相当于每样至10毫升所需的体积。例如，对于图1中的实验设计的培养物体积为60毫升</li><li>在30℃生长，振摇直到OD 600纳米 = 0.05-0.1。除去10ml的样品作为未诱导对照和0.1％阿拉伯糖添加到剩余的培养以诱导从PKM1生产四面体-YFP的。继续增长这两个非诱导和诱导文化。 1小时后，检查用荧光显微镜的诱导培养的每个单元内的单个联络点的存在（见第2节）。 </li><li>如果诱导细胞证实阻断（细胞的70％以上具有单焦点），记录OD 600nm的和通过2-D凝胶（见第3）取为分析7.5 ml样品。乘坐从非诱导控制文化的等效采样。 </li></ol></li><li>释放复制遇阻<ol><li>要释放复制块，无偿诱导无水四环素（AT）的100纳克毫升-1添加到阻止细胞10毫升的样品。继续在30℃下生长10分钟，并取为细胞密度（1毫升）中，显微镜分析（10微升）和中性中性2维琼脂糖凝胶（7.5毫升）中的样品。 </li></ol></li><li>诱导复制体崩塌<ol><li>如果细胞含有温度敏感性等位基因如dnaB TS或DNAC TS需要deactiva化，将含有受阻的细胞在烧瓶移动到42℃。取分析用试样在所需的时间点（ 例如 ，孵育1小时后）。将细胞在42℃下已培养1小时后，将细胞转移到30℃下进行10分钟（参见图1）。 注意:可以将细胞转移到30℃再启动分析。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54434/54434fig1.jpg" /> 图 1: 在FROS复制Block和释放实验步骤概述 E.携带TETO阵列大肠杆菌菌株在30℃下生长。当细胞达到上述0.05的OD 600nm的 ，四面体-YFP生产被诱导与阿拉伯糖（ARA; 0.1％）。继续增长未诱导的细胞亚群作为在30℃的控制。 2小时（见2.1） - 诱导细胞的样本后，通过1荧光显微镜进行分析。如果复制确认来将挡，取样品用于通过试管（见3.1）表示的2-D凝胶分析和生存力试验（可选）。复制堵塞可以用无水四环素（AT; 0.1微克/毫升）被移除，细胞分析10分钟后。如果细胞携带温度敏感等位基因（ 如 dnaB TS），这可以通过阻止细胞转移到42℃，适当的分析灭活。 <a href="https://www.jove.com/files/ftp_upload/54434/54434fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.荧光显微镜<ol><li>吹打500μl的熔融琼脂糖（在水中1％）到显微镜载玻片的制备琼脂糖垫;立即琼脂糖凝固之前覆盖盖玻片。直到需要在4℃下储存在湿纸巾的幻灯片（多个）。 </li><li>当琼脂糖凝固，从琼脂糖垫和吸管除去盖玻片10微升的细菌培养到垫上的中心，以防止可视期间细胞的运动。一旦样品干燥（约23分钟），更换盖玻片。将浸油下降到盖玻片并放置在光学的幻灯片。 </li><li>可视化使用相差显微镜放大100倍的细胞。 <ol><li>在成像软件中的采集对话框，设置曝光时间为100毫秒。通过选择采集捕捉图像。不要移动舞台。在Acquire对话框中，选择YFP作为关联到相机照明的外部快门。设置exporsure时间1000毫秒。关闭舞台灯光。通过选择采集捕捉荧光图像。 注意:时间可以根据所用光源，显微镜和照相机而变化。 </li><li>叠加相位和荧光图像，选择颜色从显示菜单中结合。在颜色组合对话框中，选择YFP图像作为绿色成分，并为BL相图像客户端组件。点击颜色相结合。确定是否人口已复制成功地阻止通过建立大部分细胞是否具有每单元一个焦点。 注:在视觉上识别EYFP生产的差别比较，从不加阿拉伯糖对照样本是非常有用的。 </li></ol></li></ol> 3.提取DNA /琼脂糖插头<ol><li>叠氮化钠（0.1％终浓度）从步骤1.1.3和1.2.1添加到7.5毫升培养样品。在冰上孵育至少5分钟。 注:叠氮化钠是剧毒。咨询前，开始工作的安全数据表，直到所有安全防范措施已经阅读并理解不处理它。 </li><li>离心细胞5000 xg离心10分钟以沉淀细胞。弃上清，重悬沉淀在200μlPETT四（PIV）缓冲液（10毫摩尔Tris:盐酸（pH值8.0），1M NaCl）中。微量（13，000×g离心2分钟）以沉淀细胞。迪SCARD上清液。在这个阶段，该方法粒料进一步或存储在-20℃。 </li><li>重悬在50μl的PIV和地方的最低细胞密度将细胞在50℃。对于所有的其它样品，调整PIV的卷，以便最终细胞密度为每管相同的（再悬浮在50μl 如样品1（OD 600nm处 = 0.128）;样品2（OD 600nm处 = 0.138）再悬浮于54微升）。 <ol><li>添加新鲜制备的0.8％琼脂糖溶液在PIV等体积（终浓度为0.4％; 如样品1 50微升，样品2 54微升）。确保样品，琼脂糖和PIV是50℃以上，以防止凝固。 </li></ol></li><li>治疗与40微升合适的玻璃的防水或硅酮溶液的显微镜载玻片。擦滑用纸巾，直到它是干的。 注意:此可以预先完成并经处理的载玻片存放长期在室温下。 </li><li>广场的T 20微升滴他在滑动细胞/琼脂糖悬浮液，以产生是半球形插头。 注意:第一个样品（悬浮在50微升PIV）将产生一个幻灯片5个插头。 </li><li>当插头都凝固，轻轻滑动他们入微量离心管中，并加入1 ml的细胞裂解缓冲液（10mM的Tris-HCl [pH为8]，1 M氯化钠，100mM的乙二胺四乙酸（EDTA），0​​.2％脱氧胆酸钠，0.5％ N-月桂酰肌氨酸钠盐（肌氨酸），100微克毫升- 1溶菌酶，50微克毫升- 1核糖核酸酶A）。孵育在37℃下2小时。 </li><li>除去细胞裂解缓冲液，并添加1毫升的EDTA /十二烷基肌氨酸钠/蛋白酶K（ESP）溶液（0.5M EDTA，1％N-十二烷基肌氨酸钠盐（肌氨酸），1毫克毫升- 1蛋白酶K）。在50℃下孵育过夜，或直到插头是透明的。如果需要第二过夜温育后，经过约18小时替换用新鲜的缓冲了ESP溶液。 </li><li>去除ESP缓冲液和用12ml的Tris-EDTA（TE）缓冲液（10mM的Tris-HCl，1mM EDTA中，pH值8.0）洗涤插头5次，允许30分钟的平衡与每个洗涤。在1毫升TE缓冲液保存在4℃。 </li></ol> 4.中性中性的二维凝胶电泳<ol><li>转移步骤3.8琼脂糖插头到一个新的离心管中，加入150微升限制性内切酶缓冲液（1×浓度）。加25-100单位限制酶（ 例如 ，60个单位的EcoRV的;（ 见图3A））。消化在37℃进行6-8小时。 </li><li>在4℃，倒在1倍的Tris /硼酸盐/ EDTA（TBE）0.4％琼脂糖凝胶（300ml）中成约25×25厘米的凝胶托盘。插入梳子，使孔大致相同的宽度的插头的直径。当凝胶被设置时，取出梳子和移动凝胶至室温。 </li><li>滑动消化琼脂糖塞中的一个入井中，定位插头的平整滑动靠在井其中t的侧他的DNA会进入凝胶。插入以相同的方式单个插头插入每一第二阱。 </li><li>琼脂糖移液器熔化的0.4％到井封塞的位置。 </li><li>负载1 kb的DNA梯在一个空孔，再次离开阶梯和样品之间的间隙，并在室温下过夜运行凝胶在1×TBE（16小时，55五）。 </li><li>染色在含溴化乙锭（0.3微克毫升-1）20分钟的水浴的凝胶。 注意:溴化乙锭被认为是一种有效的诱变剂和毒素。咨询前，开始工作的安全数据表，直到所有安全防范措施已经阅读并理解不处理它。 </li><li>可视化用长波紫外透射的DNA和切出各车道片段与直链，甚至切，对车道两侧最小化过量琼脂糖列入。 注意:例如，如果所关注的片段是5.5kb，切7.5厘米片段包括从5kb的DNA片段，以approximat伊利12 KB（ 见图3A）。 </li><li>放置在凝胶托盘切下的凝胶泳道在90℃对DNA的迁移方向。 注:3凝胶碎片两排可以容纳在一个25×252 厘米凝胶托盘。凝胶泳道的第一行是在所述托盘中，第二行的12.5厘米顶部。 </li><li>准备300毫升琼脂糖第二维凝胶（1X TBE缓冲液0.9％，0.3微克毫升-1溴化乙锭）。当琼脂糖冷却到50℃，吸取熔融琼脂糖围绕凝胶切片来巩固其位置。倒入剩余琼脂糖入盘的深度是与凝胶片至少水平。 </li><li>一旦凝胶固化，在含有0.3微克毫升-1溴化乙锭在220伏1×TBE electrophorese在4℃，直至该DNA已迁移大约10厘米。 注:4小时就足够了一个5.5kb的片段，但更小的片段将需要更少的时间。 </li><li>其中，消费税的基因组DNA存在ù块唱金属尺的边缘，包括它上面的凝胶，以包括不可见（ 见图3C）的DNA。 </li></ol> 5. Southern杂交<ol><li>溶液的制备<ol><li>制备脱嘌呤缓冲液（0.125 M盐酸溶液）。 注意:此缓冲器可被重新使用，并且在室温下长达1个月是稳定的。 </li><li>制备变性缓冲液（1.5 M氯化钠，0.5M氢氧化钠）。 注:变性缓冲可以一次被重新使用，并且是在室温下长达3个月稳定。 </li><li>准备中和液（1.5M氯化钠，0.5M TRIS）。调节至pH7.5，用HCl。 注意:中和缓冲区可以一次被重新使用，并且是在室温下长达3个月稳定。 </li><li>制备10倍盐水柠檬酸钠（SSC）缓冲液（0.15M的柠檬酸三钠，1.5M氯化钠，pH值是7 - 8）在步骤5.2.4和5.2.6使用。从这个缓冲区，做一个2X SSC股票（在步骤5.2.9使用）。 注:SSC 10倍和2倍SSC一重新稳定在RT 3个月。 </li><li>制备改性Church和吉尔伯特杂交缓冲液19（0.5M磷酸盐缓冲液[pH为7.2]，7％（重量/体积）十二烷基硫酸钠（SDS）的10mM EDTA）中。发表在65℃溶解使用前彻底。 </li></ol></li><li>转让<ol><li>冲洗在脱嘌呤溶液切除凝胶块在RT上的摇杆或轨道振荡器10分钟。保持搅拌速度低，以避免损坏琼脂糖块。 </li><li>简要地，用蒸馏水，然后冲洗凝胶块用变性缓冲液30分钟。简要地再次漂洗在蒸馏水中，然后孵育在中和缓冲液的凝胶块，在室温下温和搅拌30分钟。 </li><li>切断的尼龙膜对凝胶（多个）的确切大小。剪了一个缺口的右上角，以协助在未来步骤的定向膜。 注意:两个凝胶块（6个样品）可以在一个膜被可视化。 </li><li>浇足10倍SSC成TR唉，以便它不会变干过夜。创建的缓冲区的水平大约5厘米以上的平台。用浸透10倍SSC和地方平台上的3MM色谱纸3张。切割色谱纸，使得它足够长两端被浸渍在缓冲​​液和足够宽，以适应在膜上的凝胶。 </li><li>放置在饱和色谱纸的顶端的凝胶（多个）。帧用保鲜膜，以防止缓冲器绕过传送期间凝胶中的凝胶。小心地躺在凝胶（S）的顶部切膜。轻轻穿过膜滚动瓶或血清吸管除去膜和凝胶之间的气泡。 </li><li>切3MM色谱纸的三片的大小相同的膜。饱和色谱纸张与10倍SSC和放置在膜的顶部。除去已经形成的任何气泡。 </li><li>叠纸巾上吸墨水纸接着在固相载体的顶部高至少5厘米（Appro公司1公斤ximate重量为23×16cm的膜）。允许转让进行过夜。 </li><li>露出膜（DNA面朝上）于UV 1200焦耳/ m 2的以交联的DNA到膜上。之前，这一步不要冲洗。 </li><li>在2X SSC彻底冲洗膜以防止在该印迹图像高背景。立即使用印迹杂交（见5.3），或在室温下晾干，包装在塑料包装和储存在4℃。 </li></ol></li><li>杂交<ol><li>预热杂交炉和瓶子到55℃。 </li><li>加100微克毫升-1牛血清白蛋白（BSA）和10微克毫升-1非特异性DNA（ 例如鲑鱼精子DNA）到预温热杂交缓冲液。 </li><li>以允许甚至覆盖杂交缓冲液的当膜卷起时，印迹放置到尼龙的一个类似尺寸方朝上膜的DNA结合的侧啮合。卷沿其长边的印迹。滑动印迹/网杂交瓶子里。添加预热的杂交缓冲液适量展开印迹（ 如 30毫升23×16 cm 2的膜）。 </li><li>孵育在预加热的烘箱，旋转瓶，1小时。 </li><li>通过混合50纳克纯化的PCR产物同源的感兴趣的区域，的随机六聚体引物150纳克制备探针，缓冲液Klenow片段和H 2 O以使13微升的体积。煮沸3分钟，然后立即放置到冰上。加入1微升1毫米的dCTP / dGTP / dTTP的组合，在Alpha磷酸盐（α32 P-的dATP）1微升Klenow片段（5U）和50微居里脱氧腺苷5&#39;-三磷酸放射性标记的。 </li><li>孵育在室温下进行1小时。加入1 mM的dNTP混合物1微升和1微升Klenow片段。孵育20分钟在室温。 </li><li>煮沸探针5分钟，立即加入到杂交管中。在55°C杂交过夜，用旋转。 </li><li>倒出PROBE由细胞膜。 注意:探针能够被一次重复使用。 </li><li>在预温，低严格洗涤缓冲液短暂地冲洗所述膜（2×SSC，0.1％（重量/体积）SDS）。添加新鲜低严格洗涤缓冲液并在55℃用旋转孵育5分钟。重复。 </li><li>在中等严格洗涤缓冲液洗两次（1×SSC，0.1％（重量/体积）SDS）在55℃10分钟用转动。 </li><li>在高严格洗涤缓冲液洗两次（0.1×SSC，0.1％（重量/体积）SDS）中在55℃5分钟用转动。 </li><li>用纸巾除去膜和轻拍以除去过量的液体。裹保鲜膜，确保没有对保鲜膜，发生在暴露的磁带，可预先存储消隐荧光屏无残留的水分。关闭磁带和使用荧光体成像器可视化之前暴露至少2小时。 </li></ol></li></ol>

<ol>
	<li>Mirkin, E. V., Mirkin, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+stalling+at+natural+impediments.">Replication fork stalling at natural impediments.</a> Microbiol Mol Biol Rev. 71 (1), 13-35 (2007).</li><li>Gupta, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-DNA+complexes+are+the+primary+sources+of+replication+fork+pausing+in+Escherichia+coli.">Protein-DNA complexes are the primary sources of replication fork pausing in Escherichia coli.</a> Proc Natl Acad Sci U S A. 110 (18), 7252-7257 (2013).</li><li>McGlynn, P., Guy, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+forks+blocked+by+protein-DNA+complexes+have+limited+stability+in+vitro.">Replication forks blocked by protein-DNA complexes have limited stability in vitro.</a> J Mol Biol. 381 (2), 249-255 (2008).</li><li>Tanner, N. 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=Single-molecule+studies+of+fork+dynamics+in+Escherichia+coli+DNA+replication.">Single-molecule studies of fork dynamics in Escherichia coli DNA replication.</a> Nat Struct Mol Biol. 15 (2), 170-176 (2008).</li><li>Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+DNA+replication+loops+reveal+temporal+control+of+lagging-strand+synthesis.">Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.</a> Nature. 457 (7227), 336-339 (2009).</li><li>Rudolph, C. J., Upton, A. L., Lloyd, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+stalling+and+cell+cycle+arrest+in+UV-irradiated+Escherichia+coli.">Replication fork stalling and cell cycle arrest in UV-irradiated Escherichia coli.</a> Genes Dev. 21 (6), 668-681 (2007).</li><li>Jeiranian, H. A., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+replication+reduces+RecA-mediated+repair+of+UV-damaged+plasmids+introduced+into+competent+Escherichia+coli.">Inefficient replication reduces RecA-mediated repair of UV-damaged plasmids introduced into competent Escherichia coli.</a> Plasmid. 68 (2), 113-124 (2012).</li><li>Le Masson, M., Baharoglu, Z., Michel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ruvA+and+ruvB+mutants+specifically+impaired+for+replication+fork+reversal.">ruvA and ruvB mutants specifically impaired for replication fork reversal.</a> Mol Microbiol. 70 (2), 537-548 (2008).</li><li>Wang, X., Liu, X., Possoz, C., Sherratt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+Escherichia+coli+chromosome+arms+locate+to+separate+cell+halves.">The two Escherichia coli chromosome arms locate to separate cell halves.</a> Genes Dev. 20 (13), 1727-1731 (2006).</li><li>Lau, I. F., 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=Spatial+and+temporal+organization+of+replicating+Escherichia+coli+chromosomes.">Spatial and temporal organization of replicating Escherichia coli chromosomes.</a> Mol Microbiol. 49 (3), 731-743 (2003).</li><li>Gordon, G. S., 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=Chromosome+and+low+copy+plasmid+segregation+in+E.+coli:+visual+evidence+for+distinct+mechanisms.">Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms.</a> Cell. 90 (6), 1113-1121 (1997).</li><li>Possoz, C., Filipe, S. R., Grainge, I., Sherratt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+of+controlled+Escherichia+coli+replication+fork+stalling+and+restart+at+repressor-bound+DNA+in+vivo.">Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo.</a> EMBO J. 25 (11), 2596-2604 (2006).</li><li>Payne, B. T., 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=Replication+fork+blockage+by+transcription+factor-DNA+complexes+in+Escherichia+coli.">Replication fork blockage by transcription factor-DNA complexes in Escherichia coli.</a> Nucleic Acids Res. 34 (18), 5194-5202 (2006).</li><li>Brewer, B. J., Fangman, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+localization+of+replication+origins+on+ARS+plasmids+in+S.+cerevisiae.">The localization of replication origins on ARS plasmids in S. cerevisiae.</a> Cell. 51 (3), 463-471 (1987).</li><li>Jeiranian, H. A., Schalow, B. J., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+UV-induced+replication+intermediates+in+E.+coli+using+two-dimensional+agarose-gel+analysis.">Visualization of UV-induced replication intermediates in E. coli using two-dimensional agarose-gel analysis.</a> J Vis Exp. (46), (2010).</li><li>Southern, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+specific+sequences+among+DNA+fragments+separated+by+gel+electrophoresis.">Detection of specific sequences among DNA fragments separated by gel electrophoresis.</a> J Mol Biol. 98 (3), 503-517 (1975).</li><li>Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage-induced+replication+fork+regression+and+processing+in+Escherichia+coli.">DNA damage-induced replication fork regression and processing in Escherichia coli.</a> Science. 299 (5609), 1064-1067 (2003).</li><li>Mettrick, K. A., Grainge, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+blocked+replication+forks+in+vivo.">Stability of blocked replication forks in vivo.</a> Nucleic Acids Res. , (2015).</li><li>Church, G. M., Gilbert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+sequencing.">Genomic sequencing.</a> Proc Natl Acad Sci U S A. 81 (7), 1991-1995 (1984).</li><li>Michel, B., Grompone, G., Flores, M. J., Bidnenko, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+process+stalled+replication+forks.">Multiple pathways process stalled replication forks.</a> Proc Natl Acad Sci U S A. 101 (35), 12783-12788 (2004).</li><li>Michel, B., Boubakri, H., Baharoglu, Z., LeMasson, M., Lestini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination+proteins+and+rescue+of+arrested+replication+forks.">Recombination proteins and rescue of arrested replication forks.</a> DNA Repair (Amst). 6 (7), 967-980 (2007).</li><li>Carl, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+mutants+with+temperature-sensitive+synthesis+of+DNA.">Escherichia coli mutants with temperature-sensitive synthesis of DNA.</a> Mol Gen Genet. 109 (2), 107-122 (1970).</li><li>Nawotka, K. A., Huberman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+gel+electrophoretic+method+for+mapping+DNA+replicons.">Two-dimensional gel electrophoretic method for mapping DNA replicons.</a> Mol Cell Biol. 8 (4), 1408-1413 (1988).</li><li>Cole, R. S., Levitan, D., Sinden, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+psoralen+interstrand+cross-links+from+DNA+of+Escherichia+coli:+mechanism+and+genetic+control.">Removal of psoralen interstrand cross-links from DNA of Escherichia coli: mechanism and genetic control.</a> J Mol Biol. 103 (1), 39-59 (1976).</li></ol>

The authors declare that they have no competing financial interests.

During chromosome duplication, the replication machinery will encounter various impediments that prevent its progress. To ensure the entire single-origin chromosome is replicated, bacteria have numerous pathways for repair of the DNA that then enables the replisome to be reloaded20,21. Lesions, single stranded breaks, double stranded breaks and proteins tightly bound to the DNA may each be dealt with using a dedicated pathway, although there is likely to be significant overlap in these pathways. The most common impediment to replisomes in living cells is a nucleoprotein block2. However, two longstanding difficulties in studying the collision of a replisome with a nucleoprotein blockage have been how to localize the event to a known position on the chromosome and how to make the event frequent enough to allow detailed analysis. The majority of the knowledge of how E. coli copes with stalled/collapsed replication forks comes from the use of UV as a DNA damaging agent. 
The FROS protocol described here is a novel way of creating a site-specific nucleoprotein roadblock, similar to what the replisome will meet when it encounters proteins tightly bound to the chromosome during replication. The induced blockage and controlled release of this block allows the stages of DNA processing to be studied in discrete steps. The array of tetO sites used to block replication has previously been utilized to visualize chromosome organization9,10. While the array can be placed anywhere in the chromosome, for the purpose of the array described here, it should be distant from both the origin and the terminus to avoid complications in interpreting the signals observed from second rounds of initiation at the origin or from the replication fork proceeding around the opposite replichore. By optimizing the overproduction of TetR with the use of the dilute complex medium (protocol 1), we have found that sufficient TetR is produced to create a complete replication blockage that is stably maintained, while limiting the number of new rounds of replication that are initiated. The incorporation of replisome temperature sensitive mutations22 into the system ensures all replisomes are deactivated simultaneously and uncontrolled replisome reloading is prevented.
Whilst the number of foci per cell are useful for knowing the cell's replication status, the most enlightening data for DNA repair is from the analysis of the replication intermediates revealed by 2-D gel electrophoresis during an active replication blockage. Analysis of different genetic backgrounds can identify proteins involved in processing and restart of collapsed replication forks by the different patterns observed following the 2-D gel protocol. Further, intermediates from the processing of collapsed replication forks can be present at the array region or in the DNA upstream of the array, and can be visualized using 2-D gel electrophoresis. Creating duplicate agarose plugs (Protocol 3) has the advantage of allowing for digestion of the same sample with different restriction enzymes to be able to visualize replication intermediates in successive DNA regions. Therefore, the array region should be located in a position where appropriate restriction enzymes can be used to probe these regions of interest using Southern hybridization. 
The most common reasons that this technique may be unsuccessful are having either too little DNA which produces faint signals compared to background, or incomplete digestion of the DNA within the plugs. This can be caused by having too much DNA in the agarose plugs or the carry-over of some inhibitor from the lysis/washing steps. The number of cells taken for lysis within the agarose plugs may need to be varied from the protocol described here if modifications such as growth media are changed. However, it is futile to continue with the hybridization if the DNA in the plugs has not digested to completion. A repeat treatment of ESP may be required to better prepare the DNA (protocol 3.6). 
If the replication intermediates that are of interest are only transient, or are particularly rare within the cell, the DNA sample may need to be enriched for them, for example with the use of BND-cellulose which will bind short stretches of single stranded DNA, such as those found in replicating chromosomes23. Stabilization of replication intermediates by crosslinking with psoralen has also been used as a method of preserving DNA integrity24. When the intermediates are rare, it is important to optimize the probe and blocking of the membrane to prevent background signals from interfering. On completion of the blots the signals may be quantified with appropriate image processing software.
The protocol described here is an optimized system for creating a site-specific nucleoprotein blockage and analyzing replication intermediates when the replisome is unable to proceed through the blockage. With the establishment and optimization of this technique, the roles of various helicases and recombination proteins in processing the replication fork and in allowing the restart of replication can be identified. By varying the genetic background, the effect of the absence of these proteins can be analyzed. However, this technique is limited by requiring not only viable strains but strains that are also able to overproduce TetR at a rate that will create a stable nucleoprotein blockage.

在DNA复制，在复制体面临上影响其进程中的DNA的障碍。包括病变和差距DNA损伤以及异常的结构可以防止复制体从出发1。最近，人们已经发现，结合到DNA的蛋白是障碍的最常见的来源，以复制叉级数2。以下用核蛋白块的复制体的遭遇的事件的知识先前已受限于无法在已知位置来诱导在活细胞中的染色体这样的块， 在体外分析已经增强了我们的动力学行为的理解当它满足核蛋白堵塞3，以及复制体本身4,5的机械细节的活性复制体。复制的修复当前的理解通常与紫外线的损伤剂进行研究，并在体内 6-8使用质粒DNA </s了&gt;。虽然可参与DNA修复它遇到的体内核蛋白块通常从这些研究中理解后，是否有由于在复制块中的不同原因的修复途径中的分子事件的变化仍还蛋白待确定。 在这里，我们描述了一个系统，它允许在使用荧光阻遏算系统（FROS）的染色体的特定位置，以建立一个核蛋白块。我们利用大肠杆菌菌株已具有整合到染色体9 240 TETO位点的阵列的大肠杆菌 。阵列内的每个TETO站点有侧翼它通过防止在阵列内的RecA介导的重组，以增加阵列的稳定性有10 bp的随机序列。这个数组，它的变化，原本是用来理解E.大肠杆菌染色体动态10,11但随后适应preveNT 在体内的复制12。阵列已经发现稳定地保持并且当由四面体10,12结合到方框接近复制叉的100％。利用体外的类似LACO阵列已经发现少至22部位为足以阻挡90％的复制，尽管这更短的阵列是体内 13不太有效。为了适应阵列创建核蛋白堵塞，阻遏蛋白必须高度优化的条件下在那里再结合数组来创建一个路障下生产过剩。堵塞的形成，以及随后的释放，可以通过使用荧光显微镜如果使用四环素阻遏的荧光标记的变体进行监测。在每一个细胞复制的状态被看作灶的数字，其中一个焦点是指只有一个数组的副本是细胞内的存在，多灶指示复制活跃的指示。这再次活跃当核蛋白堵塞是通过添加减小四面体的操作员站点的结合亲和力充分的复制体通过阵列进行的无端诱导的逆转褶皱被启用。阻遏蛋白仍然能够结合具有足够高亲和力的DNA阵列的现在的多个拷贝可以被可视化。 在核蛋白堵塞的事件的更复杂的细节可以使用中性中性二维琼脂糖凝胶电泳和Southern杂交14-16被发现。这些技术使得DNA结构在人群的分析。那些在事件过程中形成的，并有可能在复制中间体保持未修理，可以显现。通过改变限制性内切酶和利用探头，中间体可不仅在阵列区域而且上游阵列的当复制叉退化17,18被可视化。该回归需要放置在复制体解离随后;前缘和滞后初生根丝从模板链和退火分离成彼此产生四通的DNA结构（霍利迪交界处）的模板链的同时重新退火。 使用该系统，已经表明，当它遇到该块18中的复制叉并不稳定。此外，可以利用复制体部件的温度敏感的衍生物，以防止一旦倒塌复制叉的再填装。一旦块被建立，该菌株可以转移到非许可温度下，以确保该复制体的同步失活和受控预防重装的。这种温度引起的失活可确保所有人口都折叠在给定时间内的拨叉，并允许当复制体倒塌，该DNA是如何处理的，和需要什么来重新发生什么评估启动DNA复制的过程。 这里描述的系统的一个优点是，核蛋白嵌段是完全可逆的;因此，该细胞的从核蛋白块中恢复的能力能够被遵循。加入无水四环素向细胞将缓解阻遏的紧密结合，使一个复制叉继续通过与细胞重新获得活力。堵塞的溢流可以通过中性中性二维琼脂糖凝胶电泳5分钟后进行可视化，并通过在10分钟内显微镜。此外，可行性分析可以揭示该菌株从复制堵塞恢复并继续增殖的能力。 通过改变在此处所述的实验过程中使用的菌株的遗传背景，对于这种类型的堵塞的修复途径可以阐明。

<table><tbody><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>16922</td><td>Growth media component</td></tr><tr><td>Sodium Chloride</td><td>VWR</td><td>27810.364</td><td>Growth media component</td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>92144</td><td>Growth media component</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P9791</td><td>Growth media component; Potassium buffer component</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma-Aldrich</td><td>P3786</td><td>Growth media component; Potassium buffer component</td></tr><tr><td>L-Arabinose</td><td>Sigma-Aldrich</td><td>A3256</td><td>For induction of TetR-YFP production</td></tr><tr><td>Anhydrotetracycline hydrochloride</td><td>Sigma-Aldrich</td><td>37919</td><td>Release of replication bloackage</td></tr><tr><td>Axioskop 2 Fluorescence microscope</td><td>Zeiss</td><td>452310</td><td>Visualization of cells</td></tr><tr><td>eYFP filter set</td><td>Chroma Technology</td><td>41028</td><td>Visualization of YFP</td></tr><tr><td>CCD camera</td><td>Hamamatsu</td><td>Orca-AG</td><td>Visualization of cells</td></tr><tr><td>MetaMorph&amp;nbsp; Software (Molecular Devices)</td><td>SDR Scientific</td><td>31282</td><td>Version 7.8.0.0 used in the preparation of this manuscript</td></tr><tr><td>Agarose</td><td>Bioline</td><td>BIO-41025</td><td>For agarose plugs and gel electrophoresis</td></tr><tr><td>Original Glass Water Repellent (Rain-X)</td><td>Autobarn</td><td>DIO1470</td><td>For agarose plug manufacture</td></tr><tr><td>TRIS</td><td>VWR</td><td>VWRC103157P</td><td>TE, TBE buffer component</td></tr><tr><td>Ethylene diaminetetraacetic acid</td><td>Ajax Finechem</td><td>AJA180</td><td>0.5 M EDTA disodium salt solution adjusted to pH 8.0 with NaOH.</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td>Bacteriostatic agent</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>258148</td><td>TE buffer component</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td>Cell lysis buffer component</td></tr><tr><td>N-Lauroylsarcosine sodium salt (Sarkosyl)</td><td>Sigma-Aldrich</td><td>L5125</td><td>Cell lysis and ESP buffer component</td></tr><tr><td>Rnase A</td><td>Sigma-Aldrich</td><td>R6513</td><td>Cell lysis buffer component</td></tr><tr><td>Lysozyme</td><td>Amresco</td><td>6300</td><td>Cell lysis buffer component</td></tr><tr><td>Proteinase K</td><td>Amresco</td><td>AM0706</td><td>ESP buffer component</td></tr><tr><td>Sub-Cell Model 192 Cell</td><td>BioRad</td><td>1704507</td><td>Electrophoresis system</td></tr><tr><td>UV transilluminator 2000</td><td>BioRad</td><td>1708110</td><td>Visualization of DNA</td></tr><tr><td>Ethidium Bromide</td><td>BioRad</td><td>1610433</td><td>Visualization of DNA</td></tr><tr><td>Boric acid</td><td>VWR</td><td>PROL20185.360</td><td>TBE component</td></tr><tr><td>Hybond-XL nylon memrbane</td><td>Amersham</td><td>RPN203S</td><td>Zeta-Probe Memrbane (BioRad 1620159) can also be used</td></tr><tr><td>3MM Whatman chromatography paper</td><td>GE Healthcare Life Sciences</td><td>3030690</td><td>Southern blotting</td></tr><tr><td>HL-2000 Hybrilinker</td><td>UVP</td><td>95-0031-01/02</td><td>Crosslinking of DNA and hybridization</td></tr><tr><td>Deoxyribonucleic acid from salmon sperm</td><td>Sigma-Aldrich</td><td>31149</td><td>Hybridization buffer component</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td>Denaturation buffer component</td></tr><tr><td>Trisodium citrate dihydrate</td><td>VWR</td><td>PROL27833.363</td><td>Transfer buffer</td></tr><tr><td>Sodium dodecyl sulphate (SDS)</td><td>Amresco</td><td>227</td><td>Wash buffer component</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td>Hybridization buffer component</td></tr><tr><td>Random Hexamer Primers</td><td>Bioline</td><td>BIO-38028</td><td></td></tr><tr><td>Klenow fragment</td><td>New England BioLabs</td><td>M0212L</td><td></td></tr><tr><td>dNTP Set</td><td>Bioline</td><td>BIO-39025</td><td></td></tr><tr><td>Adenosine 5&amp;rsquo;-triphosphate-&lt;sup&gt;32&lt;/sup&gt;P-ATP</td><td>PerkinElmer</td><td>BLU502A</td><td></td></tr><tr><td>Storage Phosphor Screen</td><td>GE Healthcare Life Sciences</td><td>GEHE28-9564-76</td><td>BAS-IP MS 3543 E multipurpose standard 35 cm x 43 cm screen</td></tr><tr><td>Typhoon FLA 7000</td><td>GE Healthcare Life Sciences</td><td>28-9558-09</td><td>Visualization of blot</td></tr><tr><td>Hybridization bottle</td><td>UVP</td><td>07-0194-02</td><td>35 mm x 300 mm</td></tr></tbody></table>

inducing a site specific replication blockage in e. coli using a fluorescent repressor operator system

障碍物存在于DNA，包括紧密结合的蛋白质和各种病变，会严重抑制细胞的复制机制的进程。一个复制体的失速会导致其分解从染色体，部分或全部，从而导致复制叉的崩溃。从这个崩溃的恢复是细胞准确完整的染色体复制，随后将一个必需品。所发生还原的DNA的叉，并允许复制。因此，当合拢时，细胞已经发展多种机制以高的保真度来完成。先前，在细菌中这些复制修复途径已经使用紫外线的伤害，其具有不被局限于一个已知站点的缺点进行了研究。这份手稿描述了利用荧光阻遏操作员系统（FROS）创建一个特定站点蛋白块，能够引起失速和复制的崩溃FO系统RKS在大肠杆菌 。协议的细节如何复制的状态可在单个活细胞使用荧光显微镜和DNA复制中间体被可视化可以通过2维琼脂糖凝胶电泳进行分析。复制体部件（ 例如 DnaBts）的温度敏感突变体可以被合并到系统以诱导复制叉的一个同步崩溃。此外，所涉及的这些过程的重组蛋白质和解旋酶的作用可以使用本系统内的遗传击倒进行研究。

所述FROS是诱导型，特定于站点的核蛋白块，使复制中间体在活细胞12,18被可视化。在图1中所示的一般实验设计的用于进行采样的细胞。在遗传背景的采样和变化的定时使该用于研究这样的块的修复一个通用的系统。概略说明了如何对温度敏感的突变体，如dnaB TS和DNAC TS先前已18使用时，可以在该系统中使用。 该FROS的诱导物在大肠杆菌中进行如在图1中所示的大肠杆菌菌株 用于非温度敏感菌株。要确认核蛋白块已经建立，使用后的0.1％，阿拉伯糖增长1小时荧光显微镜可视化的细胞。这个时限通常是足够的，以产生该块中的野生型菌株，但更苛刻的菌株可能需要优化。大多数细胞含有1焦点（ 图2A，+ ARA），对应于只有一个阵列的复制的细 ​​胞内，从而表明复制已被封锁。用1焦点，2灶或2个以上病灶细胞的数目在100多个细胞群进行计数。细胞的92％中发现有1焦点与2灶其余的8％。据推测，这有两个焦点间隔较开，表示该数组的两个副本的细胞，将有下一轮阻塞复制。具有两个焦点的细胞并拢表示该阵列最近被复制和核蛋白块尚未实现。 核蛋白块通过加入无水四环素（AT）与阵列的后续复制的可视化为反转具有多个病灶（ 图2A，+ ARA / AT）的细胞的累积。利用荧光观察显微术表示数组的小区内多灶已被成功复制，并有足够的时间已经过去了，让座位移开，克服任何妹妹染色体的凝聚力，这是存在。在这种情况下，加入的AT的10分钟后，将细胞的94％有2个或更多焦点而每个单元最多8灶可视化。 以确保核蛋白块确实抑制复制，细胞活力分析（ 图2B）。已经有由FROS抑制复制细胞显示在集落形成单位相比，没有在阿拉伯糖存在下生长的细胞的约1000倍的降低。这随后在此生长抑制的无水四环素显示反转的存在下生长的细胞。如果细胞不能修复后释放的DNA的核蛋白堵塞，在生存力的降低会导致。 为了显现复制中间体，细胞可以琼脂糖塞内被裂解，蛋白质和RNA除去。该DNA然后可以针对感兴趣的区域中的适当的限制酶消化。在图 3A中描绘的DNA用EcoRV这立即切断该DNA阵列之前，在阵列内和阵列后，得到5.5 kb和6.7 kb的片段在感兴趣的区域（ 图3B）中的消化。片段是理想〜3-7千对这里所描述的协议。在该范围之外的片段，可能需要使用的琼脂糖浓度和电泳条件的改变，以实现良好的分离。该DNA在第一维进行电泳和可视化（ 图3A）。如果DNA完全消化，所有车道将有均匀运行，将有高量的低分子量（小于约3Kb，这取决于限制酶）。存在于井的DNA表明不完整细胞裂解和大量的高分子量的DNA（超过10 kb的）意味着不完整的DNA消化。 在每个泳道感兴趣的DNA切下。在图3A中 ，脱氧核糖核酸以上5kb的被列入。凝胶的随后的第二维进行电泳，产生了对角线线性DNA（ 图3C）的。此凝胶中的阵列的DNA，然后通过Southern杂交（ 图3D）可视化。在畅通（ - ARA样品），两个点可以看出，对应于所述阵列的5.5 kb和6.7 ​​kb的片段，如预期。其中已复制阻断（+ ARA）的样品显示出在5.5kb的点相比的降低和增加的椭圆形的信号，指示Y形的DNA样品中的积累。该信号是定位于一个位置，标志着复制叉逮捕在整个人口有类似的地方。在加入anhydrotetracyline的，Y形的DNA信号不再能看到。最近重新启动是由整个Y型圆弧的信号的存在表明，描绘叉通过该区域迁移。 它给每个琼脂糖塞内归一的DNA，以确保样品的信号是偶数是重要的。的信号可以通过适当的图像处理软件来定量。看到另一种常见的中间是表示霍利迪路口（HJ）的形成。一个HJ被可视作为在Y弧顶圆锥信号和从线性DNA时，Y弧（ 图3D）的端部的尖峰。 <img alt="图2" src="/files/ftp_upload/54434/54434fig2.jpg" /> 图2: 荧光显微镜和VIAB<html> ility。（A）的E.大肠杆菌菌株携带TETO阵列在30℃在0.1％阿拉伯糖（+ ARA）1小时的存在下生长，然后无水四环素（AT）溶液中加入10分钟以一个亚群以释放复制块。代表性的显微照片显示了YFP生产（顶板）。比例尺= 2微米。携带1，2或2个以上病灶细胞的比例进行了列举和表示为一个百分比（下面板）。 （B）中的细胞在没有阿拉伯糖的生长（ - ARA），与阿拉伯糖（+ ARA）和与两个阿拉伯糖和无水四环素（AT; 10分钟）分别连续稀释10倍，要么斑点或涂布到仅含有琼脂氨苄青霉素（ - / + ARA样品）或无水四环素（+ AT样品氨苄青霉素）中，在30℃下生长，以确定细胞生存力。上图:代表显示板在指定稀释的增长。下图:显示平均CFU /毫升+/- SEM图。ww.jove.com/files/ftp_upload/54434/54434fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54434/54434fig3.jpg" /> 图 3:从细胞在核蛋白块DNA复制中间体的可视化 （A）的基因组DNA（1， - ARA; 2 + ARA; 3，+ ARA / AT）中裂解的琼脂糖塞内且用EcoRV限制酶消化分出在0.4％琼脂糖凝胶（55伏，16小时）。 3 KB，5 kb和10 kb的乐队表示。由白框所示的5 kb的标记上方的DNA被切除。阵列区域的EcoRV消化的（B）的示意图。输入从原点阵列复制叉被堵塞的5.5kb的片段中，当细胞已在阿拉伯糖的存在下生长。限制性位点用于通过箭头和芳指示射线被示为与线的框。 （C）中切下的DNA旋转90度，并在0.8％琼脂糖凝胶（220伏，4小时）分离。白框表示分离后的DNA切除。 （D）的阵列区域随后通过使用放射性探针阵列区Southern印迹分析显现。与该位置上的复制块电池将具有相应位于Y型弧5.5 kb的片段的信号。 <a href="https://www.jove.com/files/ftp_upload/54434/54434fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System at JoVE.com

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

诱导位点特异性复制遇阻<i&gt;电子。大肠杆菌</i&gt;用荧光阻遏操作员系统

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

inducing-site-specific-replication-blockage-e-coli-using-fluorescent

Universidad San Sebastian

Research

JoVE Journal

Biology

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

8.9K 次观看.  University of Newcastle. We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

视频: Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

科研

遗传学

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

多拷贝产质粒在抗生素耐药性演变中的作用试验

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

通过创建一个的合成Curli的操纵子工程粘附的细菌

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

生物工程

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

就地生物发光细菌细胞密度与光发射的测量与相关性研究

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

生物化学

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 ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

生物学

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

在型号蓝藻种类标记和无标记突变体的产生

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.

This protocol presents a new reporter gene system and the experimental setup to detect transcription at DNA double-strand breaks with single-molecule sensitivity.

DNA双链断裂时转录活性的活细胞成像

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far ...

诱导位点特异性复制遇阻

摘要

探索更多视频

此视频中的章节

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

多拷贝产质粒在抗生素耐药性演变中的作用试验

通过创建一个的合成Curli的操纵子工程粘附的细菌

就地生物发光细菌细胞密度与光发射的测量与相关性研究

基于荧光的感染组织细菌基因调控方法

使用延时显微镜连续测量大肠杆菌中的生物噪声

测绘细菌功能的网络和途径

单分子成像的基因调控

在型号蓝藻种类标记和无标记突变体的产生

DNA双链断裂时转录活性的活细胞成像

诱导位点特异性复制遇阻

摘要

探索更多视频

此视频中的章节

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

多拷贝产质粒在抗生素耐药性演变中的作用试验

通过创建一个的合成Curli的操纵子工程粘附的细菌

就地生物发光细菌细胞密度与光发射的测量与相关性研究

基于荧光的感染组织细菌基因调控方法

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

测绘细菌功能的网络和途径

单分子成像的基因调控

在型号蓝藻种类标记和无标记突变体的产生

DNA双链断裂时转录活性的活细胞成像

使用延时显微镜连续测量大肠杆菌中的生物噪声