S. Zhang is funded by China Scholarship Council. The authors are very grateful to former people working on polyketomycin project in this lab and Prof. Dr. Hans-Peter Fiedler, University of T&#252;bingen, for providing the polyketomycin producer. The research was supported by the DFG (RTG 1976).

1.抗菌性自然产物的鉴定<ol><li>培养继"OSMAC（一个应变许多化合物）的方法"13种不同的条件下的微生物（ 例如 ，时间，温度，pH值，介质）。选择在其中生产的化合物的观察一个介质。 </li><li> 培育链霉菌diastatochromogenesTü6028在下列条件下 <ol><li>生长在MS平板链霉菌diastatochromogenesTü6026菌株（大豆豆粉20克，D-甘露糖醇20克，MgCl 2的10mM的，琼脂1.5％，自来水1L）。接种该菌株的孢子的小环在20毫升液体TSB培养基（胰蛋白酶大豆肉汤30克，自来水1升，pH值7.2;预培养基）在锥形烧瓶中的螺旋。摇的旋转振荡器上（28℃，180转，2天）的烧瓶中。 </li><li>主要接种培养100毫升HA培养基（葡萄糖4克，酵母提取物4克，麦芽用2mL预培养的提取物10克，自来水1升，pH值7.2）。培养在28℃的应变上以180rpm的旋转摇床上6天。 </li></ol></li><li> 粗提取物的制备 <ol><li>收获细胞通过离心（10分钟，3000 XG）。 </li><li>对于下一步处理通风橱中的有机溶剂。 </li><li>为从菌丝体化合物提取，重悬细胞于丙酮的双重体积和摇在试管30分钟，180转。过滤通过商业滤纸的液体和在40℃和550巴通过旋转蒸发仪蒸发丙酮。溶解在20mL水中的提取物:乙酸乙酯（1:1）中并在180rpm下摇动它在分液漏斗中30分钟。 </li><li>为从培养介质化合物萃取，通过加入1M的盐酸调整培养液至pH 4.0。添加100ml乙酸乙酯并摇动它在分液漏斗中进行30分钟，180转。 </li><li>收集乙酸乙酯相并通过腐蒸发<html>在40℃和240巴进制蒸发。 </li></ol></li><li> 通过HPLC将粗提取物的分析 <ol><li>溶解提取物在1mL MeOH中，通过0.45μm孔径的过滤器过滤它们和运行高性能液相色谱（HPLC）14。 </li><li>在polyketomycin的情况下，装备HPLC系统带有C18前置柱（4.6×20mm的2）和C18柱（4.6×100毫米2）。使用的乙腈+ 0.5％乙酸的范围从20％至95％的线性梯度的H 2 O + 0.5％乙酸（流速:0.5毫升分钟-1）。 注:Polyketomycin具有25.9分钟的保留时间（ 见图1A）。紫外/可见光谱具有最大值在242纳米，282纳米，446纳米和最小值在262纳米和359纳米（ 见图1B）。在负作案863.2 [MH]的质量-是可检测的（ 见图1C）。 </li></ol></li></ol>"SRC ="/文件/ ftp_upload / 54952 / 54952fig1.jpg"/&gt; 图1:Polyketomycin的LC / MS分析。 （A）的HPLC色谱图6天链霉菌diastatochromogenesTü6028的培养后（λ= 430纳米）的粗提取物。 Polyketomycin具有25.9分钟（B）的紫外线的保留时间/可见光Polyketomycin的光谱。 Polyketomycin（C）的质谱中的负作案。用M / Z 863.2主峰[MH] - 。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54952/54952fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li> 使用盘扩散分析的抗菌素活性鉴定 <ol><li>接种试验菌株像革兰氏阳性菌枯草芽孢杆菌 （在LB培养基中; LB20克，自来水1升，pH7.4）和其他链霉菌菌株（在TSB培养基;胰蛋白酶大豆肉汤30克，自来水1升，pH为70.2），革兰氏阴性菌大肠杆菌 （在LB培养基中）和真菌菌株（在介质如YPD培养基;酵母提取物10g，蛋白胨20克，葡萄糖20克，自来水1升，pH7.4）中。取100微升测试应变预培养和传播他们到各自的琼脂平板上。 </li><li>溶解在甲醇（或者水或DMSO）和吸管20-50微升500微升的粗提取物或纯化的化合物到无菌纸光盘。在工作台上下，30分钟干纸盘和将它们放到与测试文化板块。准备阴性对照组（溶剂）和阳性对照（抗生素， 如安普霉素[1毫克]）。 </li><li>孵育足够的条件下板，直到试验菌株明显增长，并确定抑菌圈，如果明显。孵育大肠杆菌和芽孢杆菌 。在37℃下16小时， 链霉菌 。在28℃下2-4天，真菌菌株（主要依赖于精确检验株）一吨30℃，2天）。 </li></ol></li></ol> 2.大规模提取，纯化和结构的化合物的阐明<ol><li>培育5升HA介质S diastatochromogenes（葡萄糖4克，酵母提取物4克，麦芽提取物10g，自来水1 L，pH值7.2）。接种2毫升在含有150毫升的HA介质30×500毫升锥形瓶中的预培养物。孵育以180rpm的旋转摇床上于28℃6天的菌株。 </li><li>通过离心收集细胞（10分钟15000 XG，RT），并用乙酸乙酯提取物化合物（参见1.3节）。 </li></ol><img alt="图2" src="/files/ftp_upload/54952/54952fig2.jpg" /> 图2:工作流结构解析。该方法包括:（1）应变的培养，（2）提取，（3）纯化通过固相萃取（SPE），薄层色谱（TLC），制备型高效液相色谱（HPLC），大小排阻色谱（SEC）和（4）结构鉴定通过质量分析（MS），核磁共振（NMR）和X-射线测量。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54952/54952fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li> Polyketomycin纯化 注:纯化和结构鉴定的过程示于图2。 <ol><li>用10％的MeOH -stepwise梯度H中2 O的范围从30％至100％的MeOH，100mL的每个条件分级经C18固相萃取（SPE）柱的粗提取物。 </li><li> （1 7），为洗脱系统通过制备薄层色谱（TLC）15使用的 CH 2 Cl 2 / MeOH中进一步净化含有化合物的馏分。 </li><li>纯化通过制备型HPLC 16的化合物。装备HPLC系统带有C18前置柱（5微米，9.4×20毫米）和主柱（5微米，9.4×150毫米）。使用的乙腈+ 0.5％乙酸的H中范围从20％至95％2 O + 0.5％乙酸的梯度（流速:2.0毫升/分钟）。 </li><li>以除去溶剂和其它小的杂质，执行由尺寸排阻色谱法在MeOH 17使用柱最后纯化步骤。收集最后的纯化合物和在40℃和240巴通过旋转蒸发蒸发MeOH中。 </li></ol></li><li> 结构解析 <ol><li>溶解在600-700微升DMSO-Ð6的纯化合物（大于2毫克）（依赖于机器），记录1D NMR（1 H，13 C）和二维核磁共振（HSQC，HMBC，1 H 1小时在核磁共振谱仪18 COSY）谱。通过使用DMSO-ð6表达δ值（ppm）的化学位移。 </li><li>记录高分辨率质谱（HRESI-MS）使用高分辨率polyketomycin 19质谱仪。 </li><li>用NMR和MS数据分析10的结果的解释阐明的结构。 </li></ol></li></ol> 3.提出新的分离的化合物生物合成模式<ol><li>分析分离的化合物的结构和预测的酶，它可能参与其合成。它们分配到聚酮（I型，II或III），非核糖体多肽合成，lantipeptide，萜类化合物，或糖代谢途径8。 </li><li> 例如polyketomycin <ol><li>细分结构成明显的单部分:四环部分，两个单糖和二甲基水杨酸。 </li><li>找出，这里的部分可能来自:四环部分可能会从一聚酮化合物合酶类型派生II和甲基水杨酸部分可能通过迭代聚酮化合物合酶I型派生两个糖部分，它是6- deoxysugars ，可能是甲醇合成ZED从涉及生物合成过程中的TDP-葡萄糖-4,6-脱水酶和由两个糖基转移酶可能附着葡萄糖（ 见图3）8。 </li><li>预测集群中的推定的基因。认为这是参与单个部分的合成，在连接单元，以及在修改和剪裁分子的酶。 Polyketomycin的生物合成基因簇很可能含有编码聚酮合酶II型（因此最低的ACP，KSαUND KSβ连接扩展单元）的基因，一个迭代的聚酮合成酶I型（至少ACP，AT，KS），一TDP-葡萄糖-4,6-脱水酶（必要步骤:葡萄糖→脱氧葡萄糖）和两个糖基转移酶（附接两个糖单体的糖苷配基）8。 </li></ol></li></ol><img alt="图3" src="/files/ftp_upload/54952/54952fig3.jpg" /> 图3:Polyketomycin DIVID结构编入单添砖加瓦。 Polyketomycin由四环decaketid（PKS类型II）和二甲基水杨酸（迭代PKS I型）的，由两个脱氧糖基部分β-D- amicetose和α-L- axenose（NDP-葡萄糖-4,6-联脱水酶和需要两个糖基转移酶）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54952/54952fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4.基因组测序/采矿<ol><li> 新一代测序 <ol><li>通过序列的下一代测序技术，如Illumina公司，454焦磷酸测序或固体20的基因组DNA。对准单个读取到一个参考序列或组装从头 。 注:S. diastatochromogenesTü6028的基因组中在比勒费尔德大学的生物技术中心（CeBiTec）进行测序。所有读取被组装到基因组草图7.9兆。 </li></ol></li><li> 基因组挖掘 <ol><li>搜索开放阅读框（ORF），例如通过NCBI原核基因组注释管道21,22，RAST（使用子系统技术快速注释）23,24,25，Prokka（快速原核生物基因组注释）26或27 GenDB的使用。这些方案还提出它们的功能。在S的分析diastatochromogenes导致7000多ORF的识别Tü6028基因组草图序列。 </li><li>运行特定的BLAST（基本局部比对搜索工具）分析，以获得更多的与其它类似的基因和催化结构域28,29,30路线信息。 </li><li>对于像antiSMASH 31,32,33，NAPDOS 34和35,36 NRPSpredictor的次级代谢产物基因簇运行程序的鉴定。在链霉菌diastatochromogenes antiSMASH 31,32,33鉴定的基因组草图22田间基因簇。 </li><li>分析其酶途径（多个）推定的基因簇（聚酮化合物合酶（I型，II或III），非核糖体肽合成酶，lanthipeptide，萜类，糖代谢...）。对于含有可能参与的化合物的合成的基因簇（多个）搜索（见第3节）。在S diastatochromogenes注解簇2含有聚酮化合物合酶II型基因，三聚酮化合物合酶I型基因，一个TDP-葡萄糖-4,6-脱水酶基因和二糖基转移酶基因（见图4）。 </li><li>专注于集群中的单个基因。对于PKS I型和NRPS酰基转移酶和腺苷酰化结构域的特异性，并且因此单个延长剂单元的掺入，可以预测。也比较纳入扩展单元与分子的顺序。 antiSMASH 31,32,33还示出已知化合物的相似集群，以链接到MIBiG数据库37。 </li&gt; <li>比较这些其它化合物的化合物的结构，并检查其相似之处。 </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/54952/54952fig4.jpg" /> 图4:S. Polyketomycin生物合成基因簇和其他集群的概述antiSMASH输出diastatochromogenesTü6028。 （ 一 ）S的基因组预测生物合成基因簇的概述diastatochromogenesTü6028; （B）第2组Polyketomycin生物合成基因簇有针对性的基因。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54952/54952fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 5.生物合成基因簇的验证<ol><li>搜索与用于化合物的合成明显和重要的（必需）功能的基因。对于polyketo验证霉素基因簇的基因pokPI，编码用于从PKS II型的酮合酶的α，是由一个出帧 -deletion 的 （ 见图5B）选择并失活。可替代地，中断通过克隆的内部片段的基因（参见图5C）。 </li></ol><img alt="图5" src="/files/ftp_upload/54952/54952fig5.jpg" /> 图5:单渡线基因簇的验证。 （A）的天然基因导致功能性蛋白的翻译;该基因与内部缺失成自杀载体（B）的克隆导致导致靶基因和非功能性蛋白质的随后翻译移码的单个交叉; （C）的该基因的内部片段到自杀载体的克隆导致基因和非功能性蛋白质的随后翻译的截断。 ORIT:T已起源转让（BOT）;抗生素R:耐药性。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54952/54952fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li> 所述单交换构建体克隆 <ol><li>扩增含有pokPI通过PCR 38用引物pokPI_for和pokPI_rev的片段。 </li><li>克隆PCR产物到自杀载体pKC1132 39（安普- [R [50毫克/毫升]）。自杀载体是不能够在该霉菌菌株中复制，因此具有通过同源重组整合入靶基因，以提供安普霉素抗性。转移载体导入大肠杆菌热休克转化40克隆主机。 </li><li>隔离通过碱裂解41的载体。 </li><li>消化的片段中，削减单个切割限制酶的载体DNA。该pokPI根E，在消化 酶KPN I. </li><li>治疗与大DNA聚合酶I（Klenow酶）片段，其具有5&#39;→3&#39;聚合酶活性和3&#39;→5&#39;核酸外切活性消化的载体DNA。粘性末端的平端化和随后的再连接导致的四种碱基损失。检查这些碱通过以下步骤转化的大肠杆菌 XL1 Blue的损失，采摘单个菌落，分离质粒DNA，41及通过限制性消化和测序进一步分析。 </li></ol></li><li> 单交叉偶联构造成链霉菌 <ol><li>热休克转化40将包含pokPI基因（pKC1132_ pokPI德尔）与缺失自杀质粒导入大肠杆菌 ET12567 pUZ8002 42（卡那霉素R）。孵育在100ml LB培养基中的细胞（卡那霉素50微克毫升-1，安普霉素50微克毫升-1 </sup&gt;）过夜，在37℃。收获细胞，离心（3000 xg离心，10分钟，4℃）并通过在50毫升LB培养基中重悬洗涤细胞两次。最后，悬浮细胞在2×500μLLB培养基。 </li><li>混合500μL 大肠杆菌 pUZ8002 pKC1132_ pokPI德尔与500μL 链霉菌diastatochromogenes文化（或者用孢子）。散布在MS平板混合物（大豆豆粉20克，D-甘露糖醇20克，MgCl 2的10mM的，琼脂1.5％，自来水1L）。孵育20小时将板在28℃。 </li><li>覆盖用安普霉素（1.25毫克），并溶解在1毫升水中磷霉素（5毫克）各板，并让他们干。孵育几天将平板在28℃，直至exconjugants是可见的。 </li></ol></li><li> 检查单交换突变体 <ol><li>接种单一exconjugants在20ml TSB培养基（安普霉素50毫克毫升-1），并在28°C孵育他们三天和180转。</li><li>隔离基因组DNA和43通过PCR检查pokPI基因的中断。 </li><li>接种与验证基因中断和链霉菌野生株单克隆100毫升HA中，在28°C和180 RPM培养他们6天。提取粗提取物（参见1.3节）。检查通过HPLC-MS分析复合生产（参见1.4节）。在HPLC色谱图中相应的峰和该化合物的质量不应检测的话（参见图6）。 </li></ol></li></ol><img alt="图6" src="/files/ftp_upload/54952/54952fig6.jpg" /> 图6:S. diastatochromogenes灭活pokPI基因的HPLC分析。 S的粗提取物的HPLC色谱图（λ= 430纳米）diastatochromogenes WT（上）和突变体菌株与中断pokPI -GEN（下同）。该突变株不生产polyketomycin了。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54952/54952fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<ol>
	<li>Fleming, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+antibacterial+action+of+cultures+of+a+penicillin,+with+special+reference+to+their+use+in+isolation+of+B.+influenzae.">On the antibacterial action of cultures of a penicillin, with special reference to their use in isolation of B. influenzae.</a> Br J Exp Pathol. 10 (3), 226-236 (1929).</li><li>Lemke, A., Kiderlen, A. F., Kayser, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphotericin+B.">Amphotericin B.</a> Appl. Microbiol. Biotechnol. 68 (2), 151-162 (2005).</li><li>Kirkpatrick, P., Raja, A., LaBonte, J., Lebbos, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daptomycin.">Daptomycin.</a> Nat. Rev. Drug Discov. 2 (12), 943-944 (2003).</li><li>Zakeri, B., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+biology+of+tetracycline+antibiotics.">Chemical biology of tetracycline antibiotics.</a> Biochem. cell Biol. 86 (2), 124-136 (2008).</li><li>Schatz, A., Bugle, E., Waksman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptomycin,+a+Substance+Exhibiting+Antibiotic+Activity+Against+Gram-Positive+and+Gram-Negative+Bacteria.">Streptomycin, a Substance Exhibiting Antibiotic Activity Against Gram-Positive and Gram-Negative Bacteria.</a> Exp. Biol. Med. 55 (1), 66-69 (1944).</li><li>Burg, R. W., 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=Avermectins,+new+family+of+potent+anthelmintic+agents:+producing+organism+and+fermentation.">Avermectins, new family of potent anthelmintic agents: producing organism and fermentation.</a> Antimicrob. Agents Chemother. 15 (3), 361-367 (1979).</li><li>Egerton, J. R., Ostlind, D. 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=Avermectins,+new+family+of+potent+anthelmintic+agents:+efficacy+of+the+B1a+component.">Avermectins, new family of potent anthelmintic agents: efficacy of the B1a component.</a> Antimicrob. Agents Chemother. 15 (3), 372-378 (1979).</li><li>Walsh, C. T., Fischbach, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+products+version+2.0:+connecting+genes+to+molecules.">Natural products version 2.0: connecting genes to molecules.</a> J. Am. Chem. Soc. 132 (8), 2469-2493 (2010).</li><li>Momose, I., 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=Polyketomycin,+a+new+antibiotic+from+Streptomyces+sp.+MK277-AF1.+II.+Structure+determination.">Polyketomycin, a new antibiotic from Streptomyces sp. MK277-AF1. II. Structure determination.</a> J. Antibiot. (Tokyo). 51 (1), 26-32 (1998).</li><li>Paululat, T., Zeeck, A., Gutterer, J. M., Fiedler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+polyketomycin+produced+by+Streptomyces+diastatochromogenes.T&#252;6028.">Biosynthesis of polyketomycin produced by Streptomyces diastatochromogenes.T&#252;6028.</a> J. Antibiot. (Tokyo). 52 (2), 96-101 (1999).</li><li>Momose, I., 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=Polyketomycin,+a+new+antibiotic+from+Streptomyces.+sp.+MK277-AF1.+I.+Taxonomy,+production,+isolation,+physico-chemical+properties+and+biological+activities.">Polyketomycin, a new antibiotic from Streptomyces. sp. MK277-AF1. I. Taxonomy, production, isolation, physico-chemical properties and biological activities.</a> J. Antibiot. (Tokyo). 51 (1), 21-25 (1998).</li><li>Daum, M., 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=Organisation+of+the+biosynthetic+gene+cluster+and+tailoring+enzymes+in+the+biosynthesis+of+the+tetracyclic+quinone+glycoside+antibiotic+polyketomycin.">Organisation of the biosynthetic gene cluster and tailoring enzymes in the biosynthesis of the tetracyclic quinone glycoside antibiotic polyketomycin.</a> Chembiochem. 10 (6), 1073-1083 (2009).</li><li>Bode, H. B., Bethe, B., H&#246;fs, R., Zeeck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Big+effects+from+small+changes:+possible+ways+to+explore+nature's+chemical+diversity.">Big effects from small changes: possible ways to explore nature's chemical diversity.</a> Chembiochem. 3 (7), 619-627 (2002).</li><li>Wolfender, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC+in+Natural+Product+Analysis:+The+Detection+Issue.">HPLC in Natural Product Analysis: The Detection Issue.</a> Planta Med. 75 (07), 719-734 (2009).</li><li>Stahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thin-layer chromatography. A laboratory handbook. , (1967).</li><li>Snyder, L. R., Kirkland, J. J., Glajch, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparative+HPLC+Separation.">Preparative HPLC Separation.</a> Pract. HPLC Method Dev. , 616-642 (2012).</li><li>Granath, K. A., Kvist, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+distribution+analysis+by+gel+chromatography+on+sephadex.">Molecular weight distribution analysis by gel chromatography on sephadex.</a> J. Chromatogr. A. 28, 69-81 (1967).</li><li>Jackman, L. M., Sternhell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Nuclear+Magnetic+Resonance+Spectroscopy+in+Organic+Chemistry.">Application of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry.</a> Int. Ser. Org. Chem. , (1969).</li><li>Xian, F., Hendrickson, C. L., Marshall, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+mass+spectrometry.">High resolution mass spectrometry.</a> Anal. Chem. 84 (2), 708-719 (2012).</li><li>Metzker, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+technologies+-+the+next+generation.">Sequencing technologies - the next generation.</a> Nat. Rev. Genet. 11 (1), 31-46 (2010).</li><li>Tatusova, 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=Prokaryotic+Genome+Annotation+Pipeline.">Prokaryotic Genome Annotation Pipeline.</a> NCBI Handb. 2nd Ed. , (2013).</li><li>Angiuoli, S. V., 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=Toward+an+online+repository+of+Standard+Operating+Procedures+(SOPs)+for+(meta)genomic+annotation.">Toward an online repository of Standard Operating Procedures (SOPs) for (meta)genomic annotation.</a> OMICS. 12 (2), 137-141 (2008).</li><li>Aziz, R. 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=The+RAST+Server:+rapid+annotations+using+subsystems+technology.">The RAST Server: rapid annotations using subsystems technology.</a> BMC Genomics. 9, 75 (2008).</li><li>Overbeek, R., 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=The+SEED+and+the+Rapid+Annotation+of+microbial+genomes+using+Subsystems+Technology+(RAST).">The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST).</a> Nucleic Acids Res. 42, 206-214 (2014).</li><li>Brettin, 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=RASTtk:+a+modular+and+extensible+implementation+of+the+RAST+algorithm+for+building+custom+annotation+pipelines+and+annotating+batches+of+genomes.">RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes.</a> Sci. Rep. 5, 8365 (2015).</li><li>Seemann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prokka:+rapid+prokaryotic+genome+annotation.">Prokka: rapid prokaryotic genome annotation.</a> Bioinformatics. 30 (14), 2068-2069 (2014).</li><li>Meyer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GenDB--an+open+source+genome+annotation+system+for+prokaryote+genomes.">GenDB--an open source genome annotation system for prokaryote genomes.</a> Nucleic Acids Res. 31 (8), 2187-2195 (2003).</li><li>Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, 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=Basic+local+alignment+search+tool.">Basic local alignment search tool.</a> J. Mol. Biol. 215 (3), 403-410 (1990).</li><li>Madden, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BLAST+Sequence+Analysis+Tool.">The BLAST Sequence Analysis Tool.</a> NCBI Handb. 2nd Ed. , (2003).</li><li>Marchler-Bauer, 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=CDD:+NCBI's+conserved+domain+database.">CDD: NCBI's conserved domain database.</a> Nucleic Acids Res. 43, 222-226 (2014).</li><li>Medema, M. H., 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=antiSMASH:+rapid+identification,+annotation+and+analysis+of+secondary+metabolite+biosynthesis+gene+clusters+in+bacterial+and+fungal+genome+sequences.">antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences.</a> Nucleic Acids Res. 39, 339-346 (2011).</li><li>Blin, 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=antiSMASH+2.0--a+versatile+platform+for+genome+mining+of+secondary+metabolite+producers.">antiSMASH 2.0--a versatile platform for genome mining of secondary metabolite producers.</a> Nucleic Acids Res. 41, 204-212 (2013).</li><li>Weber, 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=antiSMASH+3.0+-+a+comprehensive+resource+for+the+genome+mining+of+biosynthetic+gene+clusters.">antiSMASH 3.0 - a comprehensive resource for the genome mining of biosynthetic gene clusters.</a> Nucleic Acids Res. 43, 237-243 (2015).</li><li>Ziemert, N., 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=The+natural+product+domain+seeker+NaPDoS:+a+phylogeny+based+bioinformatic+tool+to+classify+secondary+metabolite+gene+diversity.">The natural product domain seeker NaPDoS: a phylogeny based bioinformatic tool to classify secondary metabolite gene diversity.</a> PLoS One. 7 (3), 34064 (2012).</li><li>Rausch, C., Weber, T., Kohlbacher, O., Wohlleben, W., Huson, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+prediction+of+adenylation+domains+in+nonribosomal+peptide+synthetases+(NRPS)+using+transductive+support+vector+machines+(TSVMs).">Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs).</a> Nucleic Acids Res. 33 (18), 5799-5808 (2005).</li><li>R&#246;ttig, M., 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=NRPSSpredictor2--a+web+server+for+predicting+NRPS+adenylation+domain+specificity.">NRPSSpredictor2--a web server for predicting NRPS adenylation domain specificity.</a> Nucleic Acids Res. 39, 362-367 (2011).</li><li>Medema, M. H., 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=Minimum+Information+about+a+Biosynthetic+Gene+cluster.">Minimum Information about a Biosynthetic Gene cluster.</a> Nat. Chem. Biol. 11 (9), 625-631 (2015).</li><li>Mullis, 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=Specific+enzymatic+amplification+of+DNA+in+vitro:+the+polymerase+chain+reaction.">Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction.</a> Cold Spring Harb. Symp. Quant. Biol. 51, 263-273 (1986).</li><li>Bierman, M., 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=Plasmid+cloning+vectors+for+the+conjugal+transfer+of+DNA+from+Escherichia+coli+to+Streptomyces+spp.">Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.</a> Gene. 116 (1), 43-49 (1992).</li><li>Froger, A., Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+plasmid+DNA+into+E.+coli.using+the+heat+shock+method.">Transformation of plasmid DNA into E. coli.using the heat shock method.</a> J. Vis. Exp. (6), e253 (2007).</li><li>Bimboim, H. C., Doly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+alkaline+extraction+procedure+for+screening+recombinant+plasmid+DNA.">A rapid alkaline extraction procedure for screening recombinant plasmid DNA.</a> Nucleic Acids Res. 7 (6), 1513-1523 (1979).</li><li>MacNeil, D. J., 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=Analysis+of+Streptomyces+avermitilis.+genes+required+for+avermectin+biosynthesis+utilizing+a+novel+integration+vector.">Analysis of Streptomyces avermitilis. genes required for avermectin biosynthesis utilizing a novel integration vector.</a> Gene. 111 (1), 61-68 (1992).</li><li>Pospiech, A., Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+quick-prep+of+genomic+DNA+from+Gram-positive+bacteria.">A versatile quick-prep of genomic DNA from Gram-positive bacteria.</a> Trends Genet. 11 (6), 217-218 (1995).</li><li>Makitrynskyy, R., 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=Pleiotropic+regulatory+genes+bldA,+adpA+and+absB+are+implicated+in+production+of+phosphoglycolipid+antibiotic+moenomycin.">Pleiotropic regulatory genes bldA, adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin.</a> Open Biol. 3 (10), 130121 (2013).</li><li>Kalan, L., 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=A+cryptic+polyene+biosynthetic+gene+cluster+in+Streptomyces+calvus+is+expressed+upon+complementation+with+a+functional+bldA+gene.">A cryptic polyene biosynthetic gene cluster in Streptomyces calvus is expressed upon complementation with a functional bldA gene.</a> Chem. Biol. 20 (10), 1214-1224 (2013).</li><li>Gessner, 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=Changing+Biosynthetic+Profiles+by+Expressing+bldA+in+Streptomyces+Strains.">Changing Biosynthetic Profiles by Expressing bldA in Streptomyces Strains.</a> ChemBioChem. 16 (15), 2244-2252 (2015).</li></ol>

The authors have nothing to disclose.

在这个实验中生成的链霉菌diastatochromogenes基因组粘粒文库和多年前筛选，通过极其耗时的过程导致polyketomycin基因簇的鉴定。单个基因的表征是可能使用靶基因缺失和所得突变体12的分析。最近，获得S. diastatochromogenes的基因组序列草图允许polyketomycin生物合成基因簇的快速鉴定。我们可以很容易地检测生物合成基因，虽然基因组序列草图包含还是有很多重叠群。所述方法可以在数月内实现。但是，该过程包括许多步骤。单步骤可能会失败几次，防止发展为后续步骤: 链霉菌属是著名的，以产生生物活性化合物的能力。虽然他们往往携带超过20 BIOSynthetic基因簇，通常只有一个或两个化合物在实验室条件下产生的。在OSMAC方法（在不同条件下一株种植）以唤醒沉默的基因簇的应用程序可能有时是不够的。调节基因，如多效调节基因ADPA 44和bldA 45,46的遗传操作，也是以激活生产其它次级代谢产物的一个有效方法。 该化合物的结构的阐明， 例如 ，通过NMR分析，通常超过2毫克纯化的化合物是必要的。因此，超过10升培养物发酵通常需要。未经发酵罐，其能够维持氧气，pH值和温度条件下，它可能是在一个小的实验室来处理培养和随后的提取该金额有挑战性。在纯化过程中，该化合物可以因氧化，辐射或温度来改变。另外，将使用多个纯化步骤，较高降解的机会。 当分析所述天然产物的结构，并在基因组中的簇，有时是不容易确定合适的集群。首先，如果仅存在一个基因组序列草图集群的某些部分可能会丢失。其次，不这些所需的生物合成所有基因都在群集中。第三，有时一个簇被分成两部分由许多个碱基彼此分离。第四，它可能难以决定哪一个是相应的基因簇。在大的PKS I型或NRPS系统，它是可以计算的延长剂单元根据模块的数量的数量，或者甚至由选择域的分析识别单个延长剂单元的情况下，它变成了容易。然而，在反复工作的情况下酶的合成的化合物的预测通常是不可能的，特别是如果STRA在有40多个基因簇。第五，性质是高度复杂和充满未知化合物。通常的生物合成是不同途径的混合物。如果新的化合物尚未确定，或不相关的另一种化合物，它可能是难以确定的簇，提出一个生物合成模型并证明这一点。 一旦集群被确认，单渡线技术是验证的假设一个良好和快速的方法。 PCR，克隆到自杀载体，结合，阳性克隆的选择和生产试验是唯一需要的步骤。这种技术的一个缺点是，该载体到染色体的整合并不稳定，由于进一步的重组事件。因此，为了进一步分析单个基因时，需要精确的基因缺失。此外，它可能很难操纵在基因水平株链霉菌 。 所描述的过程可以被分配吨Ø由链霉菌菌株或其他微生物产生的任何其他化合物。有关生物合成基因簇及合成的化合物知识为我们提供了更多的机会已经修改现有的分子以改善他们反对耐药病原体斗争的目的。

从植物和微生物天然产物一直用于临床药物开发和研究的一个重要来源。第一种抗生素青霉素是由亚历山大·弗莱明1于1928年发现的真菌。现今，越来越多的天然产物在临床治疗中使用。 其生产各种次生代谢产物具有不同的生物活性的能力称为一个属是链霉菌 。 链霉菌是革兰氏阳性菌和属于类放线菌和放线菌目的。的临床上使用的抗生素几乎三分之二来自放线菌来源的，主要是从链霉菌属 ，如两性霉素2，达托霉素3或四环素4。两个诺贝尔奖被授予在链霉菌抗生素研究领域。第一个去瓦克斯曼塞尔曼链霉素的发现，第一个tibiotic抗结核病有效。 5 2015年，为诺贝尔生理学或医学奖的一部分，阿维菌素的从阿维链霉菌的发现被评为良好。阿维菌素是用于寄生虫病6,7的治疗。 为天然产物在微生物如链霉菌属发现的传统方法通常包括不同的生长条件下菌株的培养，以及提取和次生代谢产物的分析。生物活性测定（抗菌和抗癌活性如测定）被执行，以检测该化合物的活性。最后，所关注的化合物被分离和化学结构阐明。 天然产物的结构通常由它们形成的复杂分子单一部分的。有一些，但有限的，主要的生物合成途径建设集团KS，它被用于天然产物的生物合成。主要的生物合成途径是聚酮途径，通路，导致使用氨基酸萜类化合物和生物碱，通路和通路，导致糖基。各通路的特征在于一组特定酶8。基于所述化合物的结构，这些生物合成酶可以预测的。 如今，在使用下一代测序和生物信息学分析的组合的化合物的详细的结构分析可以帮助确定负责生物合成基因簇。集群信息开辟了进一步的天然产物研究的新途径。这包括异源表达来增加天然产物的基因缺失或改变，并与来自其他途径的基因组合生物合成的产量，有针对性的复合改性。 Polyketomycin被从培养液中分离独立地两种菌株， 链霉菌 。 MK277-AF1 9和链霉菌diastatochromogenesTü602810。结构通过NMR和X射线分析阐明。 Polyketomycin由四环decaketid和甲基水杨酸，由两个脱氧糖基部分β-D- amicetose和α-L- axenose相连。它显示的细胞毒性和抗菌活性，甚至对革兰氏阳性耐药株如葡萄球菌11。 生成S. diastatochromogenesTü6028的基因组粘粒图书馆和许多年前筛选。使用特定的基因探针polyketomycin基因簇的大小为52.2 kb的，含有41个基因，经过几个月的紧张工作12之后被鉴定。最近，获得S. diastatochromogenes的基因组序列草图通往polyketomycin生物合成基因簇的快速鉴定。在此概述的方法帮助IDENtify一种天然产物并阐明其生物合成基因簇进行说明，使用polyketomycin作为一个例子。 在这里，我们将解释这从天然产物其生物合成基因簇的链霉菌diastatochromogenesTü6028polyketomycin产生所示引领单一步骤。该协议包括具有抗菌性能的天然产物的鉴定和纯化。进一步的结构分析，并与从基因组采矿引线结果向生物合成基因簇的鉴定比较。此过程可以应用到从链霉菌属菌株或任何其他微生物产生的任何其他化合物。

<table border="0" cellpadding="0" cellspacing="0" width="1334">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">agar</td>
			<td style="width:397px;">Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">5210.4</td>
			<td style="width:360px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">agarose</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">6352.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-mannitol&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">4175.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glucose&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">6780.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">LB&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">X964.3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">malt extract&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">X976.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MgCl2</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">2189.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peptone&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">soy flour&#160;</td>
			<td style="width:397px;">W.Schoenemberger GmbH, Magstadt, Germany</td>
			<td style="width:231px;"></td>
			<td>Hensec-Vollsoja</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tryptic soy broth</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">X938.3</td>
			<td>Caso-Bouillon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">yeast extract&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">2363.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Solvents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Acetic acid</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">3738.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Acetone&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">9372.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Acetonitrile</td>
			<td style="width:397px;">Avantor Performance Materials B.V., Deventer, The Netherlands</td>
			<td>JT-9012-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dichlorofrom&#160;</td>
			<td>Fisher Scientific GmbH, Schwerte, Germany</td>
			<td>1530754</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">DMSO (Dimethyl sulfoxide)</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">4720.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">DMSO-d6 (Dimethyl sulfoxide-d6) 99.9atom%D</td>
			<td style="width:397px;">ARMAR Chemicals, D&#246;ttingen, Switzerland</td>
			<td style="width:231px;">15200.204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Ethyl acetate</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td>6784.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Hydrochloric acid</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">6331.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Methanol</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">7342.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">restriction enzymes</td>
			<td colspan="2">New England Biolabs GmbH, Frankfurt am Main, Germany</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">polymerase&#160;</td>
			<td colspan="2">New England Biolabs GmbH, Frankfurt am Main, Germany</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA Polymerase I Large (Klenow) Fragment</td>
			<td>Promega GmbH, Mannheim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Antibiotics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">apramycin</td>
			<td>AppliChem GmbH, Darmstadt, Germany&#160;</td>
			<td style="width:231px;">A7682.0005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">fosfomycin</td>
			<td>Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany</td>
			<td style="width:231px;">P5396-50G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">kanamycin</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td style="width:231px;">T832.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Plasmid/Vectors</td>
			<td style="width:397px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">pKC1132</td>
			<td style="width:397px;">Bierman et al. 1992</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Primer&#160;</td>
			<td style="width:397px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">pokPI_for</td>
			<td>TGATGGTGCCGCTGGCCATGG</td>
			<td rowspan="2" style="width:231px;">Primer to amplify fragment containing pokPI gene</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">pokPI_rev</td>
			<td>AGCGTTCACTGTTCCGCCCGAC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Bacterial strains</td>
			<td style="width:397px;"></td>
			<td style="width:231px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Bacillus subtilis COHN ATCC6051</td>
			<td></td>
			<td>Gram-positive test strain&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Escherichia coli ET12567 pUZ8002&#160;</td>
			<td>MacNeil et al., 1992</td>
			<td>strain for conjugation&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Escherichia coli XL1 Blue</td>
			<td style="width:397px;">Agilient Technologies, Santa Clara, USA</td>
			<td colspan="2">Gram-negative test strain + cloning host&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Streptomyces diastatochromogenes T&#252;6028</td>
			<td style="width:397px;">Paululat et al., 1999</td>
			<td>Polyketomycin producer</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Online services</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">antismash (Antibiotics and Secondary Metabolite Analysis Shell)</td>
			<td style="width:397px;">http://antismash.secondarymetabolites.org/</td>
			<td colspan="2">Detection of secondary metabolite gene cluster</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">BLAST (Basic Local Alignment Search Tool)</td>
			<td style="width:397px;">http://blast.ncbi.nlm.nih.gov/Blast.cgi</td>
			<td colspan="2">finds regions of similarity between biological sequences</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">GenDB</td>
			<td style="width:397px;">https://www.uni-giessen.de/fbz/fb08/Inst/bioinformatik/software/gendb</td>
			<td>Annotation of ORFs&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">MiBIG (Minimum Information about a Biosynthetic Gene cluster)</td>
			<td style="width:397px;">http://mibig.secondarymetabolites.org/</td>
			<td colspan="2">Database of biosyntetic gene clusters</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaPDoS</td>
			<td style="width:397px;">http://napdos.ucsd.edu/</td>
			<td colspan="2">Detection of seconary metabolite gene cluster</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">NCBI Prokaryotic Genome Annotation Pipeline</td>
			<td style="width:397px;">http://www.ncbi.nlm.nih.gov/genome/annotation_prok/</td>
			<td>Annotation of ORFs&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NRPSpredictor</td>
			<td style="width:397px;">http://nrps.informatik.uni-tuebingen.de/</td>
			<td>Detection of NRPS domains</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Prokka (rapid prokaryotic genome annotation)</td>
			<td style="width:397px;">http://www.vicbioinformatics.com/software.prokka.shtml</td>
			<td>Annotation of ORFs&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">RAST (rapid annotation using subsystems technology)</td>
			<td style="width:397px;">http://rast.nmpdr.org/</td>
			<td>Annotation of ORFs&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">other programs&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Chem Station Rev. A.09.03</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td style="width:231px;">Handling program for HPLC&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Clone Manager Suite 7</td>
			<td style="width:397px;">Scientific and Educational Software, Cary, USA</td>
			<td style="width:231px;">Designing Cloning Experiment&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Newbler v2.8</td>
			<td style="width:397px;">Roche Diagnostics</td>
			<td>Alignment of sequencing reads</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Machines&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Centrifuge Avanti J-6000, Rotor JA-10</td>
			<td>Beckman Coulter GmbH, Krefeld, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC/MS</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_Autosampler: G1313A</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_Pre-column: XBridge C18 (20 mm x 4.6 mm; Particle size: 3.5 &#181;m)</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_Column:Xbridge C18 (100 mm &#215; 4.6 mm; Particle size: 3.5 &#956;m)</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_semi-prep Pre-Column: Zorbax B-C18 (9.4 x 150 mm; Particle size: 5 &#181;m)</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_semi-prep Column: Zorbax B-C18 (9.4 x 20 mm; Particle size: 5 &#181;m)</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_Degasser: G1322A&#160;</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_Quarternary pump: G1311A</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_Diode array detector (DAD )G1315B (&#955; = 254 nm and 400 nm)</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_Quadrupole mass detector (MSD) G1946D(2-3000 m/z)</td>
			<td style="width:397px;">Agilent Technologies, Waldbronn, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rotary evaporator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_heating bath Hei-VAP Value/G3</td>
			<td colspan="2">Heidolph Instruments GmbH &#38; Co.KG, Schwabach, Germany</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">_vacuum pump system SC 920 G</td>
			<td>KNF&#160;Global Strategies&#160;AG, Sursee, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">other material&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Sephadex LH20</td>
			<td style="width:397px;">GE Healthcare,&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chromafil PVDF-45/15MS (pore size 0.45 &#181;m; filter &#216;15 mm)</td>
			<td>MACHEREY-NAGEL GmbH &#38; Co. KG, D&#252;ren, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SPE column Oasis HLB 20 35 cc (6g)</td>
			<td style="width:397px;">Waters GmbH, Eschborn, Germany&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E. coli&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td></td>
			<td>Medium: LB, Composition: LB, Amount to 1 L H2O: 20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacillus&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td></td>
			<td>Medium: LB, Composition: LB, Amount to 1 L H2O: 20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomyces sp.</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td></td>
			<td>Medium: TSB, Composition: CASO Boullion, Amount to 1 L H2O: 30 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fungus&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td></td>
			<td>Medium: YPD, Composition: Yeast extract, Amount to 1 L H2O: 10 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fungus&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td></td>
			<td>Medium: YPD, Composition: Peptone, Amount to 1 L H2O: 20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fungus&#160;</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td></td>
			<td>Medium: YPD, Composition: Glucose, Amount to 1 L H2O: 20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td>for agar plates add 2 % agar</td>
		</tr>
	</tbody>
</table>

from a natural product to its biosynthetic gene cluster: a demonstration using polyketomycin from streptomyces diastatochromogenes t&#252;6028

链霉菌菌株是众所周知的，以产生大量的具有各种生物活性的不同化合物的能力。在不同条件下培养往往导致产生新的化合物。因此，该菌株的生产培养物，用乙酸乙酯萃取，粗提取物通过HPLC进行分析。此外，提取物用于通过不同测定其生物活性进行测试。对于结构鉴定感兴趣的化合物是由不同层析方法的组合进行纯化。 加上基因组采矿基因组测序允许使用不同的计算机程序的天然产物的生物合成基因簇的鉴定。以确认正确的基因簇已被鉴定，基因失活实验必须执行。所得的突变体进行了分析用于生产特定的天然产物。一旦正确的基因簇已被灭活，该应变应失败，以产生化合物。 工作流程显示为链霉菌diastatochromogenesTü6028生产的抗菌化合物，polyketomycin。大约十年前，当基因组测序仍然是非常昂贵的，一个基因簇的克隆和鉴定是一个非常耗时的过程。快速的基因组测序基因组与联合开采加速了群识别的试验和开辟了新的方法来探索生物合成，并产生新的天然产品通过遗传学方法。本文中所描述的协议可以被分配到从链霉菌属菌株或其他微生物产生的任何其他化合物。

在此概述我们描述了从抗生素导致其生物合成基因簇的鉴定单步骤。很多年前，我们克隆粘粒库，把它们打包成噬菌体，转导大肠杆菌宿主细胞，只好筛选菌落十万来鉴定具有polyketomycin集群的重叠区域的克隆。粘粒测序也是一个困难和昂贵的过程12。 为了对菌株进行进一步的研究，我们测序链diastatochromogenesTü6028的全基因组。随着基因组序列草图，我们很容易识别polyketomycin和编码希望的化合物其他集群的生物合成基因簇。 图7比较由一个粘粒文库和煞费苦心筛选克隆识别所述生物合成簇的"旧"的方法，并且通过与一个粗略的时间尺度随后的基因组采全基因组测序的"新"方法。新的测序技术和新的基因组挖掘方案加快整个过程。 <img alt="图7" src="/files/ftp_upload/54952/54952fig7.jpg" /> 图7:"老"和"新"分配生物合成基因簇的方法比较。 "老"的方法包括用阳性克隆的选择和各粘粒（多个）的测序粘粒文库的克隆（上文）; "新"方法包括全基因组测序和-mining识别位于应变（下同）的基因组中的所有次级代谢产物基因簇。单步时间显示在一个粗略的时间尺度。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54952/54952fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes TÃƒÂ¼6028 at JoVE.com

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes T&#252;6028

这里我们介绍（一）鉴定抗菌活性的天然产物的，（B）化合物的净化，（C），其生物合成的第一种模式，（D）的基因组测序/ -mining和详细协议（ E）的生物合成基因簇的验证。

从天然产物其生物合成基因簇:一个演示使用Polyketomycin从<em&gt;链diastatochromogenes</em&gt;Tü6028

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different ...

from-natural-product-to-its-biosynthetic-gene-cluster-demonstration

Nanyang Technological University

Research

JoVE Journal

Genetics

17.1K 次观看.  Albert-Ludwigs-Universität Freiburg, Germany. 这里我们介绍（一）鉴定抗菌活性的天然产物的，（B）化合物的净化，（C），其生物合成的第一种模式，（D）的基因组测序/ -mining和详细协议（ E）的生物合成基因簇的验证。 

视频: From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies against specific peptides within the predicted sequence allow some localization experiments. However, other possible interactions with the antibodies often cannot be excluded. This situation provided an opportunity to develop a set of assays dependent on the protein sequence. Specifically, a construct containing the gene sequence coupled to the GFP coding sequence at the C-terminal end of the protein was obtained and employed for these purposes. Experiments to characterize localization, ligand affinity, and gain of function were originally designed and carried out to confirm the identification of TMEM184A as a heparin receptor1. In addition, the construct can be employed for studies addressing membrane topology questions and detailed protein-ligand interactions. The present report presents a range of experimental protocols based on the GFP-TMEM184A construct expressed in vascular cells that could easily be adapted for other novel proteins.

A construct encoding TMEM184A with a GFP tag at the carboxy-terminus designed for eukaryotic expression, was employed in assays designed to confirm the identification of TMEM184A as a heparin receptor in vascular cells.

用GFP标记TMEM184A构建肝素受体身份确认

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies ...

科研

遗传学

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

无基因克隆的基因破坏的变形链球菌菌株的生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

间歇酵母 2-混合筛序列数据的信息化分析

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

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 ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

伦蒂病毒介导基因在低附着板中制备的人类伪性沉默

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...