Name: genome-wide gene deletions in streptococcus sanguinis by high throughput pcr
Uploaded: 2012-11-23T13:00:00
Description: Watch this Scientific Journal Video about Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR at JoVE.com

这项工作是支持的补助金从美国国立卫生（PX）和R01DE018138部分，弗吉尼亚联邦大学校长科研奖励计划（PRIP）144602-3（PX）。我们感谢博士。雷震，西城区月坛窦和小静王为协助建设的全基因组突变体。我们也感谢的DNA核心设施在弗吉尼亚联邦大学进行DNA测序。

1。引物设计<ol><li>引物的设计采用了内部脚本的基础上的S.血统的 SK36基因组序列。三套引物，F1/R1，F2/R2和F3/R3被设计用于扩增的1-kb的上游序列的靶基因，APHA-3基因编码卡那霉素抗性（公里R）蛋白3和1 -kb的下游序列的靶基因，分别（ 图1）。在这些引物中，F1和R 3的设计采用ePrimer3 EMBOSS套件的方案（ <a href="http://emboss.sourceforge.net/apps/cvs/emboss/apps/index.html"...

<ol>
	<li>Sassetti, C. M., Boyd, D. H., Rubin, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+required+for+mycobacterial+growth+defined+by+high+density+mutagenesis.">Genes required for mycobacterial growth defined by high density mutagenesis.</a> Mol. Microbiol. 48, 77-84 (2003).</li><li>Kobayashi, 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=Essential+Bacillus+subtilis+genes.">Essential Bacillus subtilis genes.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 4678-4683 (2003).</li><li>Trieu-Cuot, P., Courvalin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+of+the+Streptococcus+faecalis+plasmid+gene+encoding+the+3'5"-aminoglycoside+phosphotransferase+type+III.">Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III.</a> Gene. 23, 331-341 (1983).</li><li>Paik, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+virulence+determinants+for+endocarditis+in+Streptococcus+sanguinis+by+signature-tagged+mutagenesis.">Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis.</a> Infect. Immun. 73, 6064-6074 (2005).</li><li>Lukomski, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonpolar+inactivation+of+the+hypervariable+streptococcal+inhibitor+of+complement+gene+(sic)+in+serotype+M1+Streptococcus+pyogenes+significantly+decreases+mouse+mucosal+colonization.">Nonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization.</a> Infect. Immun. 68, 535-542 (2000).</li><li>Baba, 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=Construction+of+Escherichia+coli+K-12+in-frame,+single-gene+knockout+mutants:+the+Keio+collection.">Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.</a> Mol. Syst. Biol. 2, (2006).</li><li>de Berardinis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complete+collection+of+single-gene+deletion+mutants+of+Acinetobacter+baylyi+ADP1.">A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1.</a> Mol. Syst. Biol. 4, 174 (2008).</li><li>Rodriguez, A. 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=Physiological+and+molecular+characterization+of+genetic+competence+in+Streptococcus+sanguinis.">Physiological and molecular characterization of genetic competence in Streptococcus sanguinis.</a> Mol. Oral. Microbiol. 26, 99-116 (2011).</li><li>Perry, D., Kuramitsu, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Streptococcus+mutans+Infect.">Genetic transformation of Streptococcus mutans Infect.</a> Immun. 32, 1295-1297 (1981).</li><li>Pakula, R., Walczak, W. <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+nature+of+competence+of+transformable+streptococci.">On the nature of competence of transformable streptococci.</a> J. Gen. Microbiol. 31, 125-133 (1963).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Xu, P., 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=Genome-wide+essential+gene+identification+in+Streptococcus+sanguinis.">Genome-wide essential gene identification in Streptococcus sanguinis.</a> Sci. Rep. 1, (2011).</li></ol>

为了尽量减少可能的极性的影响1与外源性抗生素基因对邻近基因的靶基因的更换，采取两个步骤，而最初的引物设计。对于大多数已删除的基因，引物R1和F3被设计用于删除后的6个碱基的30 bp的起始密码子到终止密码子之前的编码区。最后保留了30个碱基对，以保护潜在的使用相邻的下游基因的核糖体结合位点。两个相邻的基因之间的保留区域被扩展到100 bp的，当他们位于相反方向，这两种蛋白的...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;姓名</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品编号</strong</td&gt;<td<strong评论（可选）</strong</td&gt;</tr&gt;<tr&gt;<td&gt;引物F2</td&gt;<td集成DNA技术</td&gt;<td</td&gt;<td&gt; TGACTAACTAGGAGGAATAAATG<br/&gt; GCTAAAATGAGAATAT</td&gt;</tr&gt;<tr&gt;<td&gt;入门R2</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; CATTATTCCCTCCAGGTACTAAAA<br/&gt; CAATTCATCCAGT</td&gt;</tr&gt;<tr&gt;<td&gt;入门F2P</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GATAAACCCAGCGAACCATTTGA</td&gt;</tr&gt;<tr&gt;<td&gt;引物P1</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GCTTATATACCTTAGCAGGAGACA</td&gt;</tr&gt;<tr&gt;<td&gt;引物P2</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GTATGACATTGCCTTCTGCGTCC</td&gt;</tr&gt;<tr&gt;<td引物5&#39;end_R1_seq</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GCCATTTATTCCTCCTAGTTAGTCA</td&gt;</tr&gt;<tr&gt;<td引物5&#39;end_F3_seq</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GTTTTAGTACCTGGAGGGAATAATG</td&gt;</tr&gt;<tr&gt;<td引物5&#39;end_R1p_seq</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; CCTCAAATGGTTCGCTGGGTTTATC</td&gt;</tr&gt;<tr&gt;<td&gt; CSP</td&gt;<td&gt; Synprep</td&gt;<td</td&gt;<td&gt;序列：NH 2-DLRGVPNPWGWIFGR-COOH的</td&gt;</tr&gt;<tr&gt;<td&gt; DNA聚合酶</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; 11304-102</td&gt;<td&gt;铂金Taq DNA聚合酶高保真</td&gt;</tr&gt;<tr&gt;<td&gt; EcoRI位</td&gt;<td&gt;新英格兰生物实验室公司</td&gt;<td&gt; R0101S</td&gt;<td&gt;限制性内切酶</td&gt;</tr&gt;<tr&gt;<td&gt;琼脂</td&gt;<td美国生物分析</td&gt;<td&gt; AB01185</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;琼脂糖</td&gt;<td&gt; Promega公司</td&gt;<td&gt; V3125</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; BHI</td&gt;<td&gt; BD Biosciences公司</td&gt;<td&gt; 237500</td&gt;<td&gt;脑心浸液</td&gt;</tr&gt;<tr&gt;<td&gt;马血清</td&gt;<td&gt;默飞世尔科技</td&gt;<td&gt; SH3007403</td&gt;<td&gt;马血清</td&gt;</tr&gt;<tr&gt;<td&gt; KM</td&gt;<td&gt;默飞世尔科技</td&gt;<td&gt; BP906-5</td&gt;<td&gt;卡那霉素</td&gt;</tr&gt;<tr&gt;<td&gt; TH肉汤</td&gt;<td&gt; BD Biosciences公司</td&gt;<td&gt; 249240</td&gt;<td&gt; Todd Hewitt肉汤</td&gt;</tr&gt;<tr&gt;<td&gt; PureLink 96</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; K310096</td&gt;<td&gt; PureLink 96 PCR纯化试剂盒</td&gt;</tr&gt;<tr&gt;<td&gt; QIAQUICK</td&gt;<td&gt; Qiagen公司</td&gt;<td&gt; 28106</td&gt;<td&gt;的Qiaquick PCR产物纯化试剂盒</td&gt;</tr&gt;<tr&gt;<td&gt;过滤器</td&gt;<td&gt;康宁</td&gt;<td&gt; 431117</td&gt;<td&gt; 0.22微米的聚苯乙烯过滤器</td&gt;</tr&gt;<tr&gt;<td&gt; Anoxomat系统</td&gt;<td&gt;沃尔玛微生物BV</td&gt;<td&gt; Anoxomat Mark II的</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;台式离心机</td&gt;<td&gt;赛默飞世尔科技</td&gt;<td&gt; 75004377</td&gt;<tdSORVALL传奇RT Plus微孔板转子的</td&gt;</tr&gt;<tr&gt;<td&gt;凝胶</td&gt;<td&gt; UVP有限责任公司</td&gt;<td&gt; BioDoc-210</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;孵化器</td&gt;<td&gt;默飞世尔科技</td&gt;<td&gt; 11-690-625D</td&gt;<td&gt; Isotemp</td&gt;</tr&gt;<tr&gt;<tdLabnet的凝胶XL</td&gt;<td&gt; Labnet</td&gt;<td&gt; E0160</td&gt;<tdLabnet的凝胶XL</td&gt;</tr&gt;<tr&gt;<td&gt;微型离心机</td&gt;<td&gt; Eppendorf公司</td&gt;<td&gt; 22621408</td&gt;<td&gt;微量5415ŕ</td&gt;</tr&gt;<tr&gt;<td&gt;多道移液器</td&gt;<td&gt;热Scientfic</td&gt;<td&gt; 4661040，4661020</td&gt;<td&gt; Finnpipette F1</td&gt;</tr&gt;<tr&gt;<td&gt;热循环仪</td&gt;<td&gt;应用生物系统公司</td&gt;<td&gt; N805-0200</td&gt;<td&gt; GeneAmp PCR系统9700</td&gt;</tr&gt;</tbody&gt;</table

genome-wide gene deletions in streptococcus sanguinis by high throughput pcr

转座子突变和单基因缺失的两种方法适用于全基因组基因敲除细菌1,2。虽然转座子诱变是耗时更少，成本更低，并且不需要完成的基因组信息，在该方法中有两个缺点：（1）在该混合反选择竞争降低的突变体的突变体库的一个完全不同的突变体的可能性;和（2）的部分的基因失活的可能性，即基因的不完全失去的转座子的插入后，它们的功能。单基因的缺失分析可能补偿相关的缺点与转座子诱变。为了提高效率的全基因组的单基因缺失，我们尝试建立了高通量技术的使用链球菌血统作为模式生物的全基因组单基因缺失。每个基因缺失构建S.血统基因组被设计成包括1-kb的上游的靶基因，APHA-3基因，编码卡那霉素抗性的蛋白质，和1-kb的下游的靶基因。分别三套引物F1/R1 F2/R2和F3/R3，设计并合成用于PCR扩增这三个组成部分的每个删除构建在96孔板中的格式。引物R1和F3包含25-bp的序列的APHA-3基因在它们的5&#39;末端的区域是互补的。执行一次创建所有的单基因缺失构建一个大型的APHA-3基因的 PCR扩增。 APHA-3基因的启动子的初步排除卡那霉素磁带，以减少潜在的极性效应。要创建的基因缺失构建，高吞吐量的PCR扩增和纯化是在96孔板中的格式进行。线性每个基因缺失的重组PCR扩增子将通过4采用高保真DNA聚合酶的PCR反应。最初的expon差分生长阶段的S.血统 Todd Hewitt肉汤培养在补充有2.5％灭活的马血清是用来增加PCR的重组构建体的转化能力。在此条件下，高达20％的S.血统细胞可以使用〜50ng的DNA转化。根据此方法，2048个单基因缺失的突变体，最终获得的2270个基因在S.血统不包括4个基因的ORF完全包含在其他的ORF S.血统 SK36和218潜在的必需基因。该技术是高通量基因缺失结构，可以很容易使用任何变形菌的全基因组的单基因缺失。

经过PCR扩增用引物F1和R1，和F3和R3，约1 kb的上游和下游的每个S.血统基因中得到的96孔板的形式，分别为（ 图2A）。在我们的PCR条件下，使用设计引物，扩增特定产品的S.血统每个PCR反应中的基因组DNA。此结果表明，该引物对具有高度特异性的目标的S.血统 。通过PCR扩增使用F1和R3引物和3扩增子作为DNA模板，线性的重组PCR构造（〜3 kb的）的高通量的96孔板中，组成的...

Watch this Scientific Journal Video about Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR at JoVE.com

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

已经建立一个高效的全基因组单基因突变的方法使用<em&gt;链球菌血统</em作为一种模式生物。通过这种方法已经取得了高通量重组PCR和转换。

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

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Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

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