感谢麦克拉厅，拉里萨Bispo的古斯塔沃和塔瓦雷斯的技术援助。

那些生吃或熟轻食物是最关注的问题对人类的健康。例子包括蔬菜沙拉，水果，贝壳类和甲壳类动物。 1.样品采集<ol><li>采集样品，最大限度地减少污染条件下，在运输过程中保存在单独的，干净的袋子。一旦收集，样品应当存放在4℃和24小时内处理。 </li></ol> 2.富集培养制备<ol><li>水果和蔬菜: <ol><li>放置约10g材料在耐用的塑料袋中。如果在处理较大的材料，集中在水果或蔬菜的皮肤上。添加90毫升0.1M磷酸钠缓冲液，pH 7.0和过程中桨式搅拌器30秒。可替代地，该材料可手动搅动如果桨式搅拌器不可用。 </li><li>收集液体倒入两个50毫升猎鹰管。离心管在4000 XG 7分钟沉淀细菌。 </l我&gt; <li>小心取出上清液，在30ml无菌卢里亚BERTANI（LB）肉汤暂停沉淀细菌。 </li><li>孵育在振荡器上O / N，保持一个管在25℃，另一个在37℃。文化应在每分钟200振荡（OPM）被动摇。商店培养在4℃下直到需要。 </li></ol></li><li>海鲜: <ol><li>解剖出胃（牡蛎）或消化道（虾），使用无菌镊子和镊子，并放入无菌的1.5mL管中含有200微升的0.1M磷酸钠缓冲液，pH 7.0。浸渍样品以创建匀浆。 </li><li>分配5毫升LB肉汤分成两无菌5毫升管，并与海鲜匀浆接种每个。 </li><li>孵育在振荡器上O / N，保持一个管在25℃，而另一个37℃。文化应该在200 OPM动摇。商店培养在4℃下直到需要。 </li></ol></li></ol> 3.筛选的文化整合子2浓度为2.5mM。如果需要的话，洛伦茨（2011）24概述了在这个杂志的早期发行的PCR优化和故障排除方法。 <ol><li>煮DNA制备: <ol><li>分配100μl的富集培养物进入无菌0.5毫升PCR管。加热样品至99℃下进行10分钟，用水浴或加热块。卡扣冰上冷却2分钟。 </li><li>微量在14000×g离心5分钟以沉淀细胞碎片。回到冰。 DNA样品可以在-20℃冷冻直至需要的，但必须在冰上解冻。 </li></ol></li><li>筛选使用PCR 1类整合: 注:从O / N混合培养培养筛选使用HS463a / HS464 PCR协议（ 表1）1类整合。 <ol><li>解冻所有的PCR试剂在冰上，远离任何潜在SOU污染的DNA的RCE中。弥补48微升的PCR mastermix每个样品，包括阴性对照（ 表2）。免除48微升PCR主混合物中，每管使用障碍的提示。 </li><li>加入2微升的DNA解冻使用障碍的提示各PCR管。在PCR热循环仪运行相应的程序。 </li><li>为了评估扩增的结果，electrophorese 7微升PCR产物在2％琼脂糖凝胶倾和在TBE缓冲液（90毫摩尔Tris-硼酸，2mM EDTA）中运行。包括分子量标记，如一个100bp的阶梯。 </li><li>交的染色用DNA染色的凝胶，和可视化使用UV透照。检查是否有已在阴性对照无扩增。在大约470基点的任何特定样品强大的乐队表明混合培养包含携带1类整合细菌。纯粹的殖民地现在从返回的阳性PCR任何混合培养物中分离。 </li></ol></li></ol><ol><li>连续稀释和扩散板: 注意:为了分离单菌落从PCR阳性混合培养物中，混合培养物连续稀释10倍的磷酸钠缓冲液中，然后镀以回收单菌落。 <ol><li>加入1毫升混合培养至9毫升0.1M磷酸钠缓冲液，pH 7.0和通过倒置混合。加入1毫升的稀释液，以进一步9毫升磷酸盐缓冲液中，并重复直到10 -8系列稀释为止。 </li><li>蔓延100微升10 -4至10 -8稀释到LB琼脂，一式两份。孵育板O / N在用于初始混合培养相同的温度。板然后可被储存在4℃直到需要，但是最好是处理单菌落相当迅速。 </li></ol></li><li>单个菌落选择和煮DNA制备: <ol><li>从串行稀释板，本身挑单个菌落lecting许多不同菌落类型成为可能，使用的标准，如集落的大小，形状和颜色。使用无菌牙签选择从传播板单菌落。 </li><li>触摸牙签的菌落，然后转移到含有100微升无菌水PCR管。如果很大数量的菌落被筛选，然后菌落可以在微量滴定盘制备。旋转的手指之间的牙签撞出一些细胞进入水中。 </li><li>使用相同的牙签，接种LB平板与殖民地的连胜。大量菌落可以存储在标记板如果每个条纹为约1厘米长。直到选择分离适当数量的重复步骤1〜3。 </li><li>孵育LB平板O / N，在相同的温度与用于初始浓缩培养。培养物然后可被储存在4℃下直到需要。 </li><li>对于从菌悬液准备DNA，按照步骤为OUtlined 3.1节。对于纯培养裂解物的筛选，执行HS463a / HS464 PCR在3.2中列出。隔离返回一个正的PCR将用于制备基因组DNA（部分5），以及用于进一步的分析。 </li></ol></li></ol> 5.基因组DNA提取整合子阳性单菌落使用珠击25 <ol><li>接种5毫升LB肉汤与纯培养物中分离的第4.2节的样品。孵育O / N，振荡被用于初始富集培养的相同的温度。 </li><li>沉淀细胞在4000 XG 7分钟，然后取出上清。悬浮在一个裂解矩阵E快速制备管1毫升CLS-TC解决方案沉淀的细胞。使用珠打浆机，处理该样本以5.5米/秒，30秒。 </li><li>在14000×g离心5分钟离心管中并回收700微升上清液进入无菌1.5mL管中。 </li><li>加入700μl稀释1结合矩阵:5用6M关idinium硫氰酸并混合在RT 5分钟。离心机在14000 xg离心1分钟，弃去上清液。 </li><li>添加800微升盐乙醇洗涤（70％乙醇，0.1M乙酸钠）和涡流直至沉淀完全重悬。在14000×g离心洗5分钟，室温，离心1分钟，弃上清。确保所有上清液已被去除，使用微量如果必要的话，允许风干5分钟。 </li><li>通过上下吹打悬浮于200μl的TE中（10mM的Tris-HCl，1mM的EDTA，pH7.6）中的小球基质。在室温下孵育3分钟。离心机在14000×g离心3分钟，并转移160微升洗脱的DNA的到无菌管中。运行在2％w / v琼脂糖凝胶的基因组DNA提取的等分试样来检查DNA提取的得率和完整性。将纯化的基因组DNA被储存在-20℃，并需要用于PCR检测时解冻在冰上。 </li></ol> 6.诊断的PCR和DNA测序注意:在第5制备的基因组DNA将被用于所有的诊断的PCR，并以确认阳性测试为1类整合。 <ol><li>冰上解冻基因组DNA，短暂涡旋的DNA样本和脉冲旋在14000 XG为20秒。重复的PCR用HS463a / HS464（ 表1），确认该菌株是积极为471 bp的扩增（3.2节）1类整合子整合酶基因。 </li><li>阳性培养物，以种的水平鉴定是由小亚基rRNA基因（16S rDNA的）的分析来实现的。使用引物F27 / r1492（ 表1）扩增16S基因。检查采用电泳PCR产物。所有的细菌指标应生成16S扩增大约1450碱基对。 </li><li>序列的16S rRNA基因的扩增子，以确定物种的身份，但由于有可能是多个正的PCR从步骤6.2，并且许多这些阳性的将是相同的细菌种类，我们将首先区分不同物种全光照在16S PCR产物克酶切。 <ol><li>消化16S的PCR的等分试样用限制酶的Hinf 1。任何酶识别4 bp的切割位点会做，但1 的Hinf从生成目标16S良好的多样性，是一个可靠的，廉价的酶。 </li></ol></li><li>成立了30微升限制性消化mastermix（ 表2）在无菌试管，加入20微升未purfied 16S PCR产物。孵育在37℃水浴O / N。检查消化在2％琼脂糖凝胶。 </li><li>分数消化图案的Hinf所产生的1。隔离用相同的16S限制信息有可能是相同的物种。而不是每次测序16S PCR产物，序列的Hinf每1限制模式的最三名代表。 </li><li>纯化使用商业试剂盒的16S PCR产物。单链核酸外切酶试剂盒可用于，但基于列的或沉淀的方法也工作良好以除去unincorporated引物和单链DNA。序列使用引物R910（ 表1），所指定的测序设施建立反应16S扩增子。 </li><li>使用产生的DNA序列与功能BLASTN（询问NCBI数据库的DNA <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> ）。大于97％核苷酸同一性通常取为物种身份。 </li></ol> 7.映射和整合子和磁带阵列的表征注:1类整合来自人类主宰的生态系统所产生很可能是所有样品中最常见的整合。这些整合都具有最新单曲的起源，因此具有高度保守的DNA序列5。 <ol><li>一类区分1整合来自人力资源和所产生的那些自然发生环境样品中通过执行PCR引物与设置intI1F165 / intI1R476 10（ 表1）。 注:1级临床或共生菌整合将产生约300 bp的产品，而环保类1整合不会产生任何产品。 </li><li>通过放大盒阵列引物HS458 / HS459表征临床整合。该PCR将扩增intI1和3&#39;保守区段之间的区域中至多暗盒阵列的末端。因为盒的数目和身份是可变的，所述PCR产物的大小也将有所不同。 </li><li>纯化的扩增子和测​​序使用每个的扩增引物的DNA。使用序列询问使用BLASTN功能（DNA数据库<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> ）。 </li><li>确保盒的命名遵循标准化术语9，因为许多数据的证词整合基因盒■使用不一致或不明确的名称。特别注意的新基因盒，因为这些可以编码新的表型，包括基因，赋予提高传播能力，致病性或毒性8,21。 注:临床前的1类整合形式仍然散发自然的环境。特别是，那些与活性Tn的402转座子最感兴趣26。这些整合的盒的阵列将不会与引物组HS458 / HS459放大，但可以与MRG284 / MRG285引物组（ 见表1）被放大，产生可变尺寸依赖于盒式内容的扩增子。 PCR产物应纯化和DNA测序为7.2概述。 </li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Extraction+of+PCR-Competent+DNA+from+Recalcitrant+Environmental+Samples.">Rapid Extraction of PCR-Competent DNA from Recalcitrant Environmental Samples.</a> Env. Microbiol. 1096, 17-23 (2014).</li><li>Sajjad, A., Holley, M. P., Labbate, M., Stokes, H., Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+class+1+integron+with+a+complete+Tn402-like+transposition+module.">Preclinical class 1 integron with a complete Tn402-like transposition module.</a> Appl. Env. Microbiol. 77 (1), 335-337 (2011).</li><li>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=Antibiotic+resistance+in+the+environment:+A+link+to+the+clinic.">Antibiotic resistance in the environment: A link to the clinic.</a> Curr. Opinion Microbiol. 13 (5), 589-594 (2010).</li><li>Stokes, H., Nesbo, C., Holley, M., Bahl, M., Gillings, M., Boucher, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+1+integrons+predating+the+association+with+Tn402.-like+transposition+genes+are+present+in+a+sediment+microbial+community.">Class 1 integrons predating the association with Tn402.-like transposition genes are present in a sediment microbial community.</a> Journal of Bacteriology. 188, 5722-5730 (2006).</li><li>Lane, 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=.">.</a> Nucleic Acid Techniques in Bacterial Systematics. , 115-175 (1991).</li><li>Holmes, A. 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=Recombination+activity+of+a+distinctive+integron-gene+cassette+system+associated+with+stutzeri.+populations+in+soil.">Recombination activity of a distinctive integron-gene cassette system associated with stutzeri. populations in soil.</a> Journal of Bacteriology. 185, 918-928 (2003).</li></ol>

作者必须披露相关一无所获。

整合及其相关基因盒的识别可能是在预测的新机会病原体的出现，跟踪途径病原体进入人类食物链，并确定新的抗性的关键步骤和毒力决定8,21,26。本文的目的是描述筛选样品1类整合，其特征阵列盒，并确定其所在的细菌种类精简的办法。在协议中的关键步骤涉及微生物好做法，并防止污染的PCR将产生误报。 这里所描述的协议可以很容易地修改，以检测其他临床相关?...

抗生素的发现是20 世纪最伟大的科学成就之一。然而，抗生素的使用和滥用，导致对抗生素有抗药性的细菌的快速发展，而这些现在在构成21 世纪严重威胁到公众健康。细菌耐药株大多数治疗方案的兴起，引起了我们正在进入一个时代，抗菌药物不再有效1,2的可能性。 赋予抗生素抗性的遗传机制是一个古老的制度，由百万年3早于人类和抗生素的选择压力。移动遗传元件，如质粒，转座子，基因组岛，一体化接合元件和整合子可以传播抗生素抗性基因（ARG）内和细菌物种4之间。其中，整合都起到ARG传播的核心作用，尽管事实上，他们依靠质粒和转座子动员和插入到细菌基因组中5。整合捕获使用整合子整合酶基因盒，然后表达使用整合编码启动子6,7-（ 图1）盒。 整合基因盒是由单个开放阅读框（ORF），其产品能够赋予对抗生素或消毒剂8的小型移动元件。 1类整合是从临床分离5，在那里他们已经集体过130个不同的抗生素耐药基因盒9收购最常用的回收整合。 1类整合传播到人类的相关共生菌和致病菌产生含有大量的这些遗传元件10的人类废物流。据估计，10 19的细菌包含1类整合是通过污水污泥每年我发布n中的英国11。因此，毫不奇怪，1类整合赋予抗生素抗性现在正在野生鸟类，鱼类和其他野生动物12-14菌群检测。整合释放回环境构成了显著的公共健康威胁，因为收购新基因盒和复杂的重组与其他移动元素仍然存在，特别是在污水处理厂和其他水体15-18。自然环境就变成一个招兵的新的阻力因素和条件致病菌19,20。新型含整合细菌和新的ARG可以绕回到人类社会通过被污染的水和食物21,22。 ARG游戏环境监测是理解和未来23管理抗生素耐药性的关键战略。特别是，要注意食品要被生吃或轻熟，因为这些本作的新要素流动和​​病原体传播的最大威胁。 在这个协议中，用于检测，鉴定和表征1类整合并在食品及其相关基因盒一个简化的方法概述（ 图2）。使用的培养和聚合酶链反应（PCR）的组合，整合可迅速在复杂细菌群落和个体分离物检测。方法来识别细菌和整合相关基因盒的构象和身份的物种中给出。该方法适用于范围广泛的植物和动物的食物和典型的工作流程的例子给出了每个这些食物类型。

<table><tbody><tr><td>GoTaq Colourless Mastermix</td><td>Promega</td><td>M7132</td><td>Used in all PCRs</td></tr><tr><td>RNAse (Ribonuclease A from bovine pancreas)</td><td>Sigma</td><td>R6513-10MG</td><td>Used in all PCRs</td></tr><tr><td>HinFI restriction enzyme</td><td>Promega</td><td>R6201</td><td>Used to digest 16S rDNA PCR poducts. Enzyme comes with optimal buffer and BSA</td></tr><tr><td>100 bp ladder</td><td>GE Healthcare</td><td>27400701</td><td>Used as a size standard on all agarose gels</td></tr><tr><td>GelRed DNA stain</td><td>Biotium</td><td>41003</td><td>CAUTION: Personal protection must be worn when handling this material</td></tr><tr><td>Guanidinium thiocyanate</td><td>Life Technologies</td><td>AM9422</td><td>CAUTION: Personal protection must be worn when handling this material</td></tr><tr><td>CLS-TC Solution</td><td>MP Biomedicals</td><td>6540409</td><td>Resuspension solution used at the begining of the genomic DNA extraction</td></tr><tr><td>Lysing Matrix E FastPrep tubes</td><td>MP Biomedicals</td><td>116914500</td><td>Tube required for mechanical disruption of bacterial cell walls. This code is used for packs of 500 tubes, smaller quantities are available.</td></tr><tr><td>Binding matrix</td><td>MP Biomedicals</td><td>116540408</td><td>Diluted 1:5 with 6 M guanidinium thiocyanate and used in the genomic DNA extraction method.</td></tr><tr><td>Fast Prep machine</td><td>MP Biomedicals</td><td>Number of options available</td><td>MP Biomedicals has a number of FastPrep machines available to purchase. Visit &lt;a href=&quot;http://www.mpbio.com&quot; target=&quot;_blank&quot;&gt;http://www.mpbio.com&lt;/a&gt; for more information</td></tr></tbody></table>

screening foodstuffs for class 1 integrons and gene cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

混合培养物和细菌分离的intI1筛选引物组HS463a / HS464 PCR可以用来检测类整合-整合酶基因，intI1（ 图1）的存在。这个引物组可以很好地用于在混合培养物检测intI1，并且也用于筛选从扩散板（ 图2）收获的细菌菌落。阳性分离应在471碱基对使用该引物组（ 图3A）产生单一强条带。大多数阳性菌株将携带的，源自人类或他们的农业?...

Watch this Scientific Journal Video about Screening Foodstuffs for Class 1 Integrons and Gene Cassettes at JoVE.com

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

食品筛查为1类整合和基因盒

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

screening-foodstuffs-for-class-1-integrons-and-gene-cassettes

Research

JoVE Journal

Environment

9.0K 次观看.  Macquarie University. This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs. 

视频: Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

网站特定的细菌染色体工程:ΦC​​31整合介导的盒式交换（IMCE）

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

科研

生物工程

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

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

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

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

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

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

遗传学

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

位点导向 的φC31介导的疟疾 按蚊 载体中的整合和盒式交换

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

大肠杆菌中天然偶联质粒介导的水平基因转移检测

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.

This study establishes a set of standard operating procedures (SOPs) for efficiently screening and isolating intestinal bacteria capable of cleaving C-glycosides.

筛选和分离 C-糖苷裂解肠道细菌

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, ...

生物化学

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

免疫与感染

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

生物学

用于表征多药物外排系统的单拷贝基因表达

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A. baumannii is hindered by the paucity of suitable genetic manipulation tools. Here, we describe methods for utilizing a chromosomal mini-Tn7-based system to achieve single-copy gene expression in an A. baumannii strain that lacks functional RND-type efflux mechanisms. Single-copy insertion and inducible efflux pump expression are quite advantageous, as the presence of RND efflux operons on high-copy number plasmids is often poorly tolerated by bacterial cells. Moreover, incorporating recombinant mini-Tn7 expression vectors into the chromosome of a surrogate A. baumannii host with increased efflux sensitivity helps circumvent interference from other efflux pumps. This system is valuable not only for investigating uncharacterized bacterial efflux pumps but also for assessing the effectiveness of potential inhibitors targeting these pumps.

作者聚焦:使用外排缺陷型细菌菌株和单拷贝基因表达系统推进抗生素耐药性研究

We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens.

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend ...

食品筛查为1类整合和基因盒

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此视频中的章节

网站特定的细菌染色体工程:ΦC31整合介导的盒式交换（IMCE）

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

位点导向的φC31介导的疟疾按蚊载体中的整合和盒式交换

大肠杆菌中天然偶联质粒介导的水平基因转移检测

筛选和分离 C-糖苷裂解肠道细菌

沙门氏菌筛选的高通量平台.

采用共培养，以检测化学介导的种间相互作用

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

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摘要

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此视频中的章节

网站特定的细菌染色体工程:ΦC​​31整合介导的盒式交换（IMCE）

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位点导向 的φC31介导的疟疾 按蚊 载体中的整合和盒式交换

大肠杆菌中天然偶联质粒介导的水平基因转移检测

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沙门氏菌筛选的高通量平台.

采用共培养，以检测化学介导的种间相互作用

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

网站特定的细菌染色体工程:ΦC31整合介导的盒式交换（IMCE）

位点导向的φC31介导的疟疾按蚊载体中的整合和盒式交换

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统