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://jove.unicartagenaproxy.elogim.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>

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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. 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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 commo...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160; 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&#8217;-triphosphate-32P-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 35x43cm 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 x 300mm</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。 <p class="jove_content" ...

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 de Cartagena UDEC

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

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

科研

生物学

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

特定地点的重组酶（SSR）技术的原则

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

一个培养人关节软骨细胞的三维系统在滑液

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

的逻辑，实验步骤和外源天然产物生物合成的潜力，拥有复杂的抗生素红霉素通过<em&gt; E.大肠杆菌</em

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

诱导位点特异性复制遇阻

摘要

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此视频中的章节

热休克法转化到大肠杆菌质粒DNA

特定地点的重组酶（SSR）技术的原则

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌

一个培养人关节软骨细胞的三维系统在滑液

使用荧光显微镜可视化中的细菌线虫

的逻辑，实验步骤和外源天然产物生物合成的潜力，拥有复杂的抗生素红霉素通过

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

膜蛋白的绿色荧光蛋白为基础的表达筛选

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达