Name: generation of a gene-disrupted streptococcus mutans strain without gene cloning
Uploaded: 2017-10-23T14:00:00
Description: Watch this Scientific Journal Video about Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning at JoVE.com

这项工作得到了日本促进科学协会的支持 [授予数字 16K15860 T.M. 和15K15777 新罕布什尔州]。

1. 入门设计 <ol> <li> 准备2步融合 PCR 的引物和基因破坏的验证. 
	注意: 表 1 显示了本协议中使用的入门序列。 图 1 显示了用于生成 变形链球菌 和 #916 的2步融合 PCR 方法的示意图; gtfC 。
	<ol> <li> 设计用于在基因组中替换目标区域的霉抗性基因 ( spc r ) 7 。此协议将 gtfC 基因的整个?...

<ol>
	<li>Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hybrid+genes+without+the+use+of+restriction+enzymes:+gene+splicing+by+overlap+extension.">Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.</a> Gene. 77 (1), 61-68 (1989).</li><li>Horton, R. M., Cai, Z., Ho, S. M., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+splicing+by+overlap+extension:+tailor-made+genes+using+the+polymerase+chain+reaction.">Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.</a> BioTechniques. 54 (3), 129-133 (2013).</li><li>Kuwayama, 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=PCR-mediated+generation+of+a+gene+disruption+construct+without+the+use+of+DNA+ligase+and+plasmid+vectors.">PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors.</a> Nucleic Acids Res. 30 (2), E2 (2002).</li><li>Sakurai, I., Mizusawa, N., Wada, H., Sato, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digalactosyldiacylglycerol+is+required+for+stabilization+of+the+oxygen-evolving+complex+in+photosystem+II.">Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II.</a> Plant Physiol. 145 (4), 1361-1370 (2007).</li><li>Aoki, H., Shiroza, T., Hayakawa, M., Sato, S., 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=Cloning+of+a+Streptococcus+mutans+glucosyltransferase+gene+coding+for+insoluble+glucan+synthesis.">Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis.</a> Infect Immun. 53 (3), 587-594 (1986).</li><li>Hanada, N., 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=Isolation+and+characterization+of+the+Streptococcus+mutans+gtfC+gene,+coding+for+synthesis+of+both+soluble+and+insoluble+glucans.">Isolation and characterization of the Streptococcus mutans gtfC gene, coding for synthesis of both soluble and insoluble glucans.</a> Infect Immun. 56 (8), 1999-2005 (1988).</li><li>LeBlanc, D. J., Lee, L. N., Inamine, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+nucleotide+base+sequence+analysis+of+a+spectinomycin+adenyltransferase+AAD(9)+determinant+from+Enterococcus+faecalis.">Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis.</a> Antimicrob Agents Chemother. 35 (9), 1804-1810 (1991).</li><li>Macrina, F. L., Tobian, J. A., Jones, K. R., Evans, R. P., Clewell, D. 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+cloning+vector+able+to+replicate+in+Escherichia+coli+and+Streptococcus+sanguis.">A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis.</a> Gene. 19 (3), 345-353 (1982).</li><li>Murata, T., Hanada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+chloride+channel+permease+to+fluoride+resistance+in+Streptococcus+mutans.">Contribution of chloride channel permease to fluoride resistance in Streptococcus mutans.</a> FEMS Microbiol Lett. 363 (11), (2016).</li><li>Perry, D., Wondrack, L. M., 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+putative+cariogenic+properties+in+Streptococcus+mutans.">Genetic transformation of putative cariogenic properties in Streptococcus mutans.</a> Infect Immun. 41 (2), 722-727 (1983).</li><li>Lau, P. C., Sung, C. K., Lee, J. H., Morrison, D. A., Cvitkovitch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+ligation+mutagenesis+in+transformable+streptococci:+application+and+efficiency.">PCR ligation mutagenesis in transformable streptococci: application and efficiency.</a> J Microbiol Methods. 49 (2), 193-205 (2002).</li><li>Ge, X., Xu, P. <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+gene+deletions+in+Streptococcus+sanguinis+by+high+throughput+PCR.">Genome-wide gene deletions in Streptococcus sanguinis by high throughput PCR.</a> J Vis Exp. (69), (2012).</li></ol>

在本议定书中, 1st PCR 的引物必须被设计为在基因组中放大大约 1 kb 的目标区域的上游和下游侧翼区域。这样长的侧翼序列对于提高同源重组的效率是必要的。
本协议中的引物序列 (向上反转、下向前、 spcr前向和spcr反转) 都是根据该中断构造的合并站点自动确定的。每个引物包括15基地补充到 2nd PCR 模板在 5 ' 地区的结束。更长的...

基因功能分析通常涉及一种表型比较的野生类型菌株和应变, 其中一个特定的基因的利益已被打乱。这种基因破坏技术已被用于基因功能分析的各种分类群, 从细菌到哺乳动物。基因中断的构造是产生基因中断的菌株所必需的;这种脱氧核糖核酸 (脱氧核糖核酸) 构造包括与目标基因的上游和下游侧翼区域融合的抗生素抗性标记基因, 并通过同源重组将其纳入基因组。使用含有抗生素的选择性培养基可以分离出基因紊乱的菌株。传统的方法用于构建的基因中断的应变需要复杂的步骤, 如放大的目标基因的聚合酶链反应 (PCR), 结扎基因成合适的质粒, 消化的限制酶, 并 religation 插入抗生素抗性标记基因。这个过程很耗时, 需要几天到几个星期, 取决于每个步骤的成功与否。此外, 破坏构造的设计也依赖于靶基因的限制性酶位点。为了解决这些问题, pcr 的 DNA 剪接方法1,2为设计基因中断的变种人网柄3和Synechocystis sp. PCC68034已报告。在本研究中, 利用一种改进的 pcr 方法 (指定的2步融合 PCR 技术) 来建立一个以相对快速、简便和廉价的方式产生基因中断的链球菌菌株的方法。遗传转化的构造和电穿孔。
2步融合 pcr 需要基因组 DNA, 抗生素抗性标记基因作为 pcr 模板, 和四对适当设计的寡核苷酸引物。在第一步 (1st pcr), 目标基因的一个 1 kb 的上游侧翼区域, 目标基因的一个 1 kb 的下游侧翼区域和抗生素抗性标记基因通过 pcr 放大。反向底漆和前向引物的5个区域分别为上游和下游侧翼区域的放大, 包括与标记基因末端互补的15碱基。标记基因扩增的前、反向引物的5个区域, 类似地包括目标基因上游侧翼区域的下端和目标基因下游侧翼区域的上端的15基。分别.因此, 三放大的片段在融合点包含重叠的30基对区域。在第二步 (2nd pcr) 中, 采用嵌套 pcr 引物进行 pcr, 使用 1st pcr 作为模板放大的三片段。模板的互补区域通过 2nd PCR 相互连接。最后, 采用电穿孔法对野生型菌株进行同源重组的构造。成功的基因破坏可以通过 PCR 方法通过特异的引物来验证。虽然破坏结构使用高保真 dna 聚合酶放大, 但额外的酶反应, 如 dna 结扎或 dna 消化, 是不需要生成的结构。此外, 在基因组上的一个被删除或保留的区域可以根据底漆的设计灵活地选择。
在目前的工作中, 删除的整个编码区域的gtfC基因, 编码葡萄糖在变形链球菌, 是为了证明快速, 容易的基因破坏方法的链球菌物种。此外, 以相同的方式生成了gtfB中断的应变。由gtfC和gtfB编码的 glucosyltransferases 有助于龋牙科生物膜的开发5,6。对野生型菌株的生物膜形成能力 (变形量)、 gtfC-被破坏的应变 (变形者δgtfC) 和gtfB中断的应变 (变形者δgtfB) 进行了评估用于比较表型分析。这个协议扩展了 two-step 方法的应用为基因中断对一个另外的种类和可能被修改对基因功能的研究在更大的种类分类群。

<table>
	<tbody>
		<tr>
			<td>Streptococus mutans</td>
			<td>Stock strain in the lab.</td>
			<td></td>
			<td>Wild-type strain (Strain UA159)</td>
		</tr>
		<tr>
			<td>Genomic DNA extraction kit</td>
			<td>Zymo Research</td>
			<td>D6005</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead-beating disruption apparatus</td>
			<td>Taitec</td>
			<td>GM-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic genes</td>
			<td>Eurofins Genomics</td>
			<td>Custom-ordered</td>
			<td>Spectinomycin- and erythromycin-resistance gene</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Astec</td>
			<td>PC-708</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Eurofins Genomics</td>
			<td>Custom-ordered</td>
			<td>35 bases in length</td>
		</tr>
		<tr>
			<td>DNA polymerase</td>
			<td>Takara</td>
			<td>R045A</td>
			<td>High-fidelity DNA polymerase</td>
		</tr>
		<tr>
			<td>Gel extraction kit</td>
			<td>Nippon Genetics</td>
			<td>FG-91202</td>
			<td>DNA extraction from agarose gel</td>
		</tr>
		<tr>
			<td>Gel band cutter</td>
			<td>Nippon Genetics</td>
			<td>FG-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Beckman Coulter</td>
			<td>DU 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bio-rad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation cuvette</td>
			<td>Bio-rad</td>
			<td>1652086</td>
			<td>0.2-cm gap</td>
		</tr>
		<tr>
			<td>Anaeropack</td>
			<td>Mitsubishi Gas Chemical</td>
			<td>A-03</td>
			<td>Anaerobic culture system</td>
		</tr>
		<tr>
			<td>Brain heart infusion broth</td>
			<td>Becton, Dickinson</td>
			<td>237500</td>
			<td>Bacterial culture media</td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Wako</td>
			<td>195-11531</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Wako</td>
			<td>057-07151</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako</td>
			<td>196-00015</td>
			<td>For biofilm development</td>
		</tr>
		<tr>
			<td>CBB R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td>For biofilm staining</td>
		</tr>
	</tbody>
</table>

generation of a gene-disrupted streptococcus mutans strain without gene cloning

典型的解释特定基因的功能的方法包括对野生型菌株和一种被破坏的基因的应变的比较表性分析。基因破坏 DNA 结构包含一个合适的抗生素抗性标记基因是有用的产生基因中断菌株的细菌。然而, 传统的建设方法, 需要基因克隆步骤, 涉及复杂和耗时的协议。在这里, 一个相对简便, 快速, 和 cost-effective 的方法为目标基因的破坏在变形链球菌描述。该方法利用2步融合聚合酶链反应 (PCR) 产生的破坏结构和电穿孔的遗传转化。这种方法不需要酶反应, 除了 PCR, 并提供更大的灵活性, 从设计的破坏结构。电穿孔的使用有利于制备合格的细胞, 提高转化效率。本方法可适用于不同品种的基因阻断菌株的生成。

图 2显示了 1st PCR 中的每个产品的大小, 如在1% 琼脂糖凝胶上观察到的, 与预测的大约 1 kb 的大小一致。图 3显示了 2nd PCR 产物的凝胶电泳分析。图 4显示了由破坏构造转化而来的变形人菌落, 以及菌落 PCR 产物的凝胶电泳分析。所有殖民地的增都是预测的大小。 <strong class...

Watch this Scientific Journal Video about Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning at JoVE.com

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

我们描述了一种简便, 快速, 和相对廉价的方法, 产生基因中断的变形链球菌菌株;这种技术可以适应于各种物种的基因中断菌株的产生。

无基因克隆的基因破坏的变形链球菌菌株的生成

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

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