Name: transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination
Uploaded: 2012-10-08T12:00:00
Description: Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com

我要感谢奥尔加·德索萨·席尔瓦的技术援助。这项工作是由瑞士国家科学基金会（SNSF）31003A_127029格兰特的支持。

的TransFLP方法（ 图1）通过三种不同的方法可以举出：Ⅰ）的两个相邻的基因，编码毒力决定因素五删除霍乱 （ 例如 ，ΔctxAB）;Ⅱ）去除的致病岛（ 例如 ，ΔVPI-1）和III）的T7 RNA聚合酶-依赖型启动子序列的融合利益一个基因的上游，这随后允许其人工表达的各菌株背景（ 例如，T7-tfoX）（ 图2）。 1。制备甲壳素片<...

<ol>
	<li>Miller, V. L., Mekalanos, J. 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+novel+suicide+vector+and+its+use+in+construction+of+insertion+mutations:+osmoregulation+of+outer+membrane+proteins+and+virulence+determinants+in+Vibrio+cholerae+requires+toxR.">A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR.</a> J. Bacteriol. 170, 2575-2583 (1988).</li><li>Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P., Kushner, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+for+generating+deletions+and+gene+replacements+in+Escherichia+coli.">New method for generating deletions and gene replacements in Escherichia coli.</a> J. Bacteriol. 171, 4617-4622 (1989).</li><li>Kato, C., Ohmiya, R., Mizuno, T. <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+method+for+disrupting+genes+in+the+Escherichia+coli+genome.">A rapid method for disrupting genes in the Escherichia coli genome.</a> Biosci. Biotechnol. Biochem. 62, 1826-1829 (1998).</li><li>Donnenberg, M. S., Kaper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+an+eae+deletion+mutant+of+enteropathogenic+Escherichia+coli+by+using+a+positive-selection+suicide+vector.">Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector.</a> Infect. Immun. 59, 4310-4317 (1991).</li><li>De Souza Silva, O., Blokesch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+Vibrio+cholerae+by+combining+natural+transformation+with+FLP+recombination.">Genetic manipulation of Vibrio cholerae by combining natural transformation with FLP recombination.</a> Plasmid. 64, 186-195 (2010).</li><li>Meibom, K. L., Blokesch, M., Dolganov, N. A., Wu, C. -. Y., Schoolnik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitin+induces+natural+competence+in+Vibrio+cholerae.">Chitin induces natural competence in Vibrio cholerae.</a> Science. 310, 1824-1827 (2005).</li><li>Pollack-Berti, A., Wollenberg, M. S., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+transformation+of+Vibrio+fischeri+requires+tfoX+and+tfoY.">Natural transformation of Vibrio fischeri requires tfoX and tfoY.</a> Environ. Microbiol. 12, 2302-2311 (2010).</li><li>Marvig, R. L., Blokesch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+transformation+of+Vibrio+cholerae+as+a+tool-optimizing+the+procedure.">Natural transformation of Vibrio cholerae as a tool-optimizing the procedure.</a> BMC Microbiol. 10, 155 (2010).</li><li>Blokesch, M., Schoolnik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serogroup+Conversion+of+Vibrio+cholerae+in+Aquatic+Reservoirs.">Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs.</a> PLoS Pathog. 3, e81 (2007).</li><li>Cherepanov, P. P., Wackernagel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+disruption+in+Escherichia+coli:+TcR+and+KmR+cassettes+with+the+option+of+Flp-catalyzed+excision+of+the+antibiotic-resistance+determinant.">Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant.</a> Gene. 158, 9-14 (1995).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T., Ford, N., Nolan, C., Ferguson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A laboratory manual. 1, (1989).</li><li>Reyes-Lamothe, R., Possoz, C., Danilova, O., Sherratt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+positioning+and+action+of+Escherichia+coli+replisomes+in+live+cells.">Independent positioning and action of Escherichia coli replisomes in live cells.</a> Cell. 133, 90-102 (2008).</li><li>Karaolis, D. K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Vibrio+cholerae+pathogenicity+island+associated+with+epidemic+and+pandemic+strains.">A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains.</a> Proc. Natl. Acad. Sci. USA. 95, 3134-3139 (1998).</li><li>Ikeda, R. A., Ligman, C. M., Warshamana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T7+promoter+contacts+essential+for+promoter+activity+in+vivo.">T7 promoter contacts essential for promoter activity in vivo.</a> Nucleic Acids Res. 20, 2517-2524 (1992).</li><li>Pan, S. H., Malcolm, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+background+expression+and+improved+plasmid+stability+with+pET+vectors+in+BL21+(DE3).">Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3).</a> Biotechniques. 29, 1234-1238 (2000).</li><li>Faruque, S. M., Zhu, J., Asadulghani, M., Kamruzzaman, J. J., Mekalanos, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+diverse+toxin-coregulated+pilus-positive+Vibrio+cholerae+strains+fails+to+demonstrate+evidence+for+Vibrio+pathogenicity+island+phage.">Examination of diverse toxin-coregulated pilus-positive Vibrio cholerae strains fails to demonstrate evidence for Vibrio pathogenicity island phage.</a> Infect. Immun. 71, 2993-2999 (2003).</li><li>Joelsson, A., Liu, Z., Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+phenotypic+diversity+of+quorum-sensing+systems+in+clinical+and+environmental+isolates+of+Vibrio+cholerae.">Genetic and phenotypic diversity of quorum-sensing systems in clinical and environmental isolates of Vibrio cholerae.</a> Infect. Immun. 74, 1141-1147 (2006).</li><li>Heidelberg, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequence+of+both+chromosomes+of+the+cholera+pathogen+Vibrio+cholerae.">DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae.</a> Nature. 406, 477-483 (2000).</li></ol>

的TransFLP上述方法和其他地方已被广泛使用在我们的实验室。种遗传操作是可行的，包括其中包括：删除单个基因和基因簇的基因岛（ 例如 ，VPI-1），缺失，插入感兴趣的基因的上游侧（ 例如 ，启动子序列）的序列，并插入序列结束时的一个特定的基因（ 例如 ，编码的亲和标签）。 一个导入的先决条件TransFLP是，各自的五霍乱菌株是自然变形。?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>甲壳素片</td><td>西格玛</td><td> C9213 </td><td>高压灭菌要求</td></tr><tr><td>可选：Micropulser（Flp重组PBR-FLP编码的情况下，抗生素盒应该被切除） </td><td> Biorad公司</td><td> 165-2100 </td><td>或类似的electroporators </td></tr></tbody></table>

transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination

有几种方法可用来操纵细菌染色体1-3。这些协议大多依靠的条件复制型质粒插入（ 例如 PIR依赖或对温度敏感的复制窝藏1,2）。这些质粒基于同源性的介导的重组整合到细菌染色体中。这样的插入突变体往往直接用于实验的设置。可替换地，可以进行选择质粒切除后跟其损失为革兰氏阴性菌，往往依赖于计数器可选择的果聚糖蔗糖酶的酶编码的sacB基因的 4。的切除可以恢复的插入前的基因型或在经修饰的基因的染色体和质粒编码的副本之间的交换的结果。这种技术的一个缺点是，它是耗时的。首先被克隆的质粒，它需要五的水平转移到霍乱 （最多n个<html> otably通过交配与E.大肠杆菌供体菌株）或人工转变后者;和切除该质粒是随机的，可以恢复初始的基因型或创建所需的修改，如果没有施加正选择的。在这里，我们提出了一个方法，为快速操作的五霍乱弧菌染色体（）5（ 图1）。此TransFLP方法是基于最近发现的几丁质的自然能力介导的诱导在此有机体6和其他五，如弧菌属代表鲵 7。自然能力允许的游离DNA的摄取，包括PCR产生的DNA片段。一旦占用，存在一个最小的250-500的bp的侧翼同源区域8给出的染色体DNA的重组。包括选择标记这些侧翼区之间可以方便地检测经常发生转化的。 T“&gt;这种方法可以用于不同的遗传操作的霍乱弧菌 ，可能还有其他的自然胜任的细菌。我们提供了三个新的例子可以用这种方法来完成的，除了我们之前发表的研究单基因缺失和另外的亲和标签序列。优化步骤，初始协议的几丁质引起的自然转换6注册成立在此TransFLP的协议，包括（其中包括）更换螃蟹壳碎片市售甲壳素片，捐赠PCR衍生的DNA为转化材料9，和另外的FLP重组目标网站（FRT）5。FRT收网站允许位点定向切除10 Flp重组酶介导的的选择标记。

代表性的结果在图4至图6中所示的三个例子的。第一种方法的目的是在删除邻近的基因CTXA的和 ctxB。他们一起进行编码的主要致病因子V。霍乱弧菌 ，霍乱毒素。我们设计寡核苷酸为TransFLP方法，如上述那样，按照图3。中间窝藏FRT-侧翼于原始的DNA位点的ctxAB和最终删除ctxAB和释放的抗性盒的菌株的抗生素抗性盒的菌株的亲本菌株，均测试其基因型...

Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com 

TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination (Video) | JoVE 

一个快速的方法来修改的基因组<em&gt; V.霍乱</em&gt;进行说明。这些修改包括删除单个基因，基因簇和基因组岛屿以及集成的短序列（如启动子元件或亲和标签序列）。该方法是基于对自然的改造和FLP重组。

TransFLP - A基因修改方法<em&gt;霍乱弧菌</em基于自然转化和FLP重组

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

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Immunology and Infection

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

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