Name: generation of a gene-disrupted streptococcus mutans strain without gene cloning
Uploaded: 2017-10-23T14:00:00.000+00:00
Duration: 12 min 6 s
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 基因的整个编码区域替换为 spc r 。
		<li> 在5和 #39 中包括与 "spc" r 的上、下两端互补的15基, 分别为上反转和下向引物的区域。同样, 还包括与 gtfC 的上游侧翼区域的下端以及在5和 #39 上的 gtfC 的下游侧翼区域的上端互补的15基地; spc r 前向和 spc 的区域 -分别反转底漆 ( 图 1A )。
		注意: 上反转、下向前、 spc r 前向和 spc r 反向底漆的顺序将根据中断构造的合并站点自动确定. </li>
		<li> 设计向上和向下反转引物以构成和 #62; gtfC 基因的上游和下游侧翼区域分别为 1 kb。根据上反和下向引物的熔化温度分别设置前向和向下反转底漆的熔化温度 ( T m ) ( 图 1A ). 
		注意: 请参阅以下公式以确定 T m : T m (and #176; C) = 2 (* NA + * NT) + 4 (* NC + NG)-5, 其中 ** 表示具有指定标识 (A、T、C 或 G) 的入门核苷酸的数量. </li>
		<li> 设计 2 nd PCR ( 图 1B ) 的嵌套底漆 (N_up 前向和 N_up). 
		注: 虽然最外层的底漆对 (向前和向下反转) 可用作 2 nd PCR 的引物, 但在本协议中设计了嵌套底漆 (N_up 和 N_up 反转)。2 nd PCR 通常需要嵌套的底漆, 详见 代表性结果 。
		<li> 设计特定于 spc r 的底漆。该引物用于菌落 PCR 筛选 gtfC 中断. </li>
		<li> 设计用于验证 gtfC 中断的底漆. 
		注: 使用这些引物扩增的 PCR 产品横跨 gtfC 或 spc r 和 gtfC 的上游或下游侧翼区域之间的边界。入门集和 #39; gtfC 前向和 gtfC 上-反向和 #39; 和 #39; gtfC 下向前和 gtfC 向下反转和 #39; 特定于 gtfC 和 #39; gtfC 前向和 spc r 2-反转和 #39; 和 #39; spc r 2 向前和 gtfC 下反转和 #39; spc r 的特定项。
	</ol> </li> </ol> 2。基因组 DNA 从 s. 变形 <ol> <li> 条纹储存牡丹 s UA159 到脑心输液 (BHI) 琼脂板上。在厌氧条件下, 在37和 #176 的夜间孵育盘. </li>
	<li> 用一根消毒过的牙签拿起一个菌落, 将它接种到5毫升的 BHI 汤中。将 s. 牡丹 的区域性一夜之间孵化为37和 #176; C 在厌氧条件下. </li>
	<li> 在2000和 #215 离心细菌细胞培养15分钟; 重5毫升磷酸盐缓冲盐水 (PBS) 中的细胞颗粒. </li>
	<li> 在2000和 #215 离心15分钟; 重200和 #181 中的细胞小球; PBS 的 L. </li>
	<li> 根据制造商提供的说明从悬浮液中提取基因组 dna, 使用珠 beating-base 基因组 dna 提取试剂盒. 
	<ol> <li> 将悬浮液传输到包含玻璃珠的2.0 毫升样品管 (包括在套件中)。在细胞悬浮液中加入750和 #181; L 溶解溶液 (包括在试剂盒中). </li>
		<li> 盖紧并将管子对称地放在磁珠跳动的破坏装置的管架上。在最高速度为5分钟的过程。在1万和 #215 离心1分钟的微珠样品; g. 将上清液转移到旋转过滤器 (包括在套件中) 在2.0 毫升收集管和离心机为 1 min 在7000和 #215; g . </li>
		<li> 添加1200和 #181; L 的 DNA 结合缓冲 (包括在工具箱中) 到滤液。将800和 #181 的混合物转到自旋柱 (包括在套件中), 以2.0 毫升的收集管和离心机1分钟, 在1万和 #215; g. </li>
		<li> 丢弃流式, 添加200和 #181; l 清洗缓冲器 (包括在套件中), 以洗涤柱矩阵, 离心1分钟, 在1万和 #215; g. 丢弃流, 添加500和 #181; 洗涤缓冲器 (包括在套件中) 的旋转柱洗柱矩阵, 离心1分钟, 在1万和 #215; g. </li>
		<li> 将自旋列放入新的 1.5 mL 离心管中, 并将100和 #181 的洗脱缓冲器 (包括在套件中) 添加到柱形矩阵中. </li>
		<li> 离心三十年代在1万和 #215; g 洗基因组 DNA。通过测量 260 nm 和 280 nmusing 分光光度计的吸光度来估计 DNA 浓度和纯度, 并确认 260 / 280 比率大于 1.8. </li>
	</ol> </li> </ol> 3。pcr 放大 <ol> <li> 执行 1 st pcr 使用牡丹 s 基因组和合成的 spc r 基因作为 pcr 模板 (参见 表 1 )。使用上面描述的三套引物 ( 图 1A ), 放大 gtfC 基因的上游侧翼区域、 gtfC 基因的下游侧翼区域以及 spc r 基因, 按 表 2 和 表 3 . 
	注: PCR 引物、试剂和扩增周期分别在 表 1 、 表 2 和 表 3 中进行了总结. </li>
	<li> 在1% 琼脂糖凝胶上分级每个 PCR 产品, 并使用凝胶带切割器对相应的频段进行切除. </li>
	<li> 使用自旋柱和硅胶膜凝胶萃取试剂盒从凝胶中纯化每个放大的 DNA 产物. 
	<ol> <li> 将凝胶切片转换为干净的离心管, 并将其与500和 #181; L 绑定缓冲区 (包括在套件中)。将混合物孵育在55和 #176; C 为15分钟, 直到凝胶切片完全溶解. </li>
		<li> 将自旋列 (包括在套件中) 放入收集管 (包括在套件中), 将样品加载到自旋柱上, 并在1.1万和 #215 上离心三十年代; g 。放弃流动, 添加600和 #181; 洗涤缓冲器 (包括在套件中), 以洗涤硅胶膜, 并离心三十年代在1.1万和 #215; g . </li>
		<li> 在1.1万和 #215 丢弃流经和离心机2分钟; g 干燥二氧化硅膜. </li>
		<li> 将自旋列放入一个新的 1.5 mL 离心管中, 添加50和 #181; L 洗脱缓冲液 (包括在套件中), 室温孵育2分钟. </li>
		<li> 在1.1万和 #215 离心2分钟; g 洗放大的 DNA。通过测量 260 nm 和 280 nmusing 分光光度计的吸光度来估计 DNA 浓度和纯度, 并确认 260 / 280 比率大于 1.8. </li>
	</ol> </li> <li> 使用三近似摩尔片段作为 pcr 模板执行2步融合 pcr 的 2 nd pcr, 按 表 2/st荣 > 和 表 3 . 
	注意: 这些片段被放大和拼接使用嵌套的引物和 #39; N_up 向前和 N_down-反向和 #39; 或最外层的底漆和 #39; 向上和向下反转和 #39; ( 图 1B )。PCR 引物、试剂和扩增周期分别在 表 1 、 表 2 和 表 3 中进行了总结. </li>
	<li> 在0.8% 琼脂糖凝胶上加载1/10 的 PCR 反应混合物 (5 和 #181; L), 并将放大的带大小与预期的带大小进行比较, 以确认适当的增 ( 图 1C ) 的生成. </li>
	<li> 将剩余的最终 PCR 产物浓缩为无纯化的乙醇沉淀. 
	<ol> <li> 添加55和 #181; 超纯水到剩余的 PCR 混合物调整总容量到100和 #181; l. 添加1/10 卷的3米醋酸钠 (10 和 #181; l), pH 5.2, 其次是2.5 量的100% 乙醇 (250 和 #181; l)。在-80 和 #176 中混合和冻结30分钟; C. </li>
		<li> 离心20分钟, 在1.5万和 #215; g 在4和 #176; C, 检查管底部的颗粒, 并丢弃上清液。用1毫升70% 乙醇洗涤颗粒, 离心5分钟, 在1.5万和 #215; g 在4和 #176; C, 并丢弃上清。空气干燥颗粒约30分钟, 溶解与10和 #181; 超纯水的 L. </li>
	</ol> </li> </ol> 4。合格的细胞制备和细胞转化 <ol> <li> 条纹股票 牡丹 s UA159 到 BHI 琼脂板上。在厌氧条件下, 在37和 #176 的夜间孵育盘. </li>
	<li> 用一根消毒过的牙签拿起一个菌落, 将它接种到5毫升的 BHI 汤中。将 s. 牡丹 的区域性一夜之间孵化为37和 #176; C 在厌氧条件下. </li>
	<li> 将2毫升的 S. 牡丹 s 培养到50毫升的 BHI 汤, 并在37和 #176 孵育6-8 小时; C 到光学密度在600毫微米 (OD 600 ) 0.2-0.4 在厌氧条件下. </li>
	<li> 在2000和 #215 离心细胞15分钟; g at 4 和 #176; C、丢弃上清, 然后用40毫升的 ice-cold 水冲洗两次细胞, 然后40毫升10% 的甘油. 
	注: 残余物质会影响随后的电穿孔. </li>
	<li> 用巴斯德吸管将上清液仔细吸出, 因为在10% 甘油中, 细胞团的粘附量减少了。重1毫升新鲜10% 甘油, 免除50和 #181; L 悬浮入每1.5 毫升离心管, 并且存放在-80 和 #176; C 直到使用前. </li>
	<li> 将50和 #181 混合在一起; l 分 ice-cold 5 和 #181 的合格细胞; 上文3节所述的破坏结构 l。将混合物添加到电穿孔试管 (电极之间的距离为0.2 厘米)。采用 金黄色葡萄球菌 模式 (1.8 伏、600、#937、10、#181、1脉) 的电穿孔装置 electroporate. </li>
	<li> 将500和 #181 中的单元格挂起, 并在电穿孔后立即 BHI 肉汤, 并添加50和 #181; 悬浮物的 l 霉 BHI 琼脂板. 
	注: 电穿孔后额外的孵育时间不需要。但是, 在电穿孔后1-2 小时的孵化可以提高转化效率. </li>
	<li> 在37和 #176 下孵育盘2-6 天; C. 在厌氧条件下. 
	注意: S 变形 和 #916; gtfB 是按照上面的描述构造的。将 gtfB 基因的整个编码区域替换为 变形链球菌 和 #916 中的红霉素抵抗基因 8 ; gtfB. 每个 S. 变形菌 菌株在37和 #176 的 BHI 汤中培养; C. 在厌氧条件下. </li>
</ol> 5。基因中断和存储的验证 <ol> <li> 选取随机菌落并将其接种到 pcr 混合物中, 以按 表 2 和 表 3 进行菌落 pcr。PCR 引物、试剂和扩增周期分别在 表 1 、 表 2 和 表 3 中进行了总结. </li>
	<li> 在1% 琼脂糖凝胶上加载 PCR 反应混合物以确认 spc r 特定的 DNA 带。
	<li> 在10毫升含霉的 BHI 汤中, 用无菌牙签和亚文化细胞选择阳性菌落, 2 天在37和 #176; C 在厌氧条件下. </li>
	<li> 验证 S. 变形 和 #916 的生成; gtfC 
	<ol> <li> 平均划分单元格挂起。从上面描述的细胞中提取基因组 DNA (步骤 2.3. 2.5.5.)。以基因组 DNA 为 pcr 模板, 使用引物来验证 gtfC 中断 ( 表 1 ) 按 表 2 和 表 3 . 
		注: PCR 引物、试剂和扩增周期分别在 表 1 、 表 2 和 表 3 中进行了总结. </li>
		<li> 在2% 琼脂糖凝胶上加载 PCR 混合物, 并将放大的带大小与预期的带大小进行比较, 以确认适当增的生成 . </li>
		<li> 将剩余的电池悬浮液离心15分钟, 在2000和 #215; g at 4 和 #176; c. 重5毫升的 PBS 中的细胞团. </li>
		<li> 离心15分钟, 在2000和 #215; g at 4 和 #176; c. 重1.5 毫升的 BHI 汤中含有25% 甘油的细胞团, 贮存于-80 和 #176; 林-20 和 #176; c. </li>
	</ol> </li> </ol> 6。表型分析 <ol> <li> 将每个 的牡丹 s 的应变置于 BHI 琼脂板上。在厌氧条件下, 在37和 #176 的夜间孵育盘. </li>
	<li> 使用无菌牙签取一个单一的菌落, 并将其接种到2毫升的 BHI 汤中, 或没有适当的抗生素。在37和 #176 的夜间孵育; C. 在厌氧条件下. </li>
	<li> 接种20和 #181; 夜间养殖的 L 在玻璃试管中含有1% 或5% 蔗糖, 不含抗生素的 BHI 肉汤; 在无氧条件下, 在37和 #176 的情况下, 在一夜之间培养与试管在倾斜位置的细胞;
	<li> 涡流玻璃试管十年代和醒酒文化悬浮。用蒸馏水冲洗试管三次。添加1毫升的0.25% 马斯亮辉煌蓝色 (CBB), 以染色的生物膜在管壁上, 然后孵化1分钟. </li>
	<li> 醒酒染色液, 用蒸馏水冲洗试管三次, 并烘干试管. </li>
	<li> 以视觉方式评估 CBB 染色生物膜的颜色密度和延伸度, 以确定表型分析中的生物膜形成能力. </li>
</ol>

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作者没有什么可透露的。

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>&lt;em&gt;Streptococus mutans&lt;/em&gt;</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

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 ...

generation-gene-disrupted-streptococcus-mutans-strain-without-gene

Research

JoVE Journal

Genetics

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

7.6K 次观看.  Tsurumi University School of Dental Medicine. 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。

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

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

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods.
Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound.
The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes T&#252;6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.

Here we present a detailed protocol of (A) the identification of a natural product with antibiotic activity, (B) the purification of the compound, (C) the first model of its biosynthesis, (D) genome sequencing/-mining and the (E) verification of the biosynthetic gene cluster.

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 ...

科研

遗传学

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

生物学

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

链球菌中高分子质量蛋白的纯化

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

免疫与感染

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

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

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

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

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 ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

在伪单体中生成帧内基因删除突变体，并在简单小鼠感染模型中测试病毒衰减

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

无标记基因删除由絮凝血盒等位基因交换突变在衣原体沙眼

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

在血清型 1 型肺炎链球菌 菌株 519/43 中构建突变体

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

的产生<em&gt;肠杆菌</em&gt; SP。 YSU营养缺陷型使用转座子突变

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

在型号蓝藻种类标记和无标记突变体的产生

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

无基因克隆的基因破坏的变形链球菌菌株的生成

摘要

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此视频中的章节

从天然产物其生物合成基因簇:一个演示使用Polyketomycin从

绿色怪物承载多基因缺失的酵母菌株的产生过程

链球菌中高分子质量蛋白的纯化

全基因组基因缺失

在伪单体中生成帧内基因删除突变体，并在简单小鼠感染模型中测试病毒衰减

无标记基因删除由絮凝血盒等位基因交换突变在衣原体沙眼

在血清型 1 型肺炎链球菌菌株 519/43 中构建突变体

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

的产生

在型号蓝藻种类标记和无标记突变体的产生

无基因克隆的基因破坏的变形链球菌菌株的生成

摘要

探索更多视频

此视频中的章节

从天然产物其生物合成基因簇:一个演示使用Polyketomycin从

绿色怪物承载多基因缺失的酵母菌株的产生过程

链球菌中高分子质量蛋白的纯化

全基因组基因缺失

在伪单体中生成帧内基因删除突变体，并在简单小鼠感染模型中测试病毒衰减

无标记基因删除由絮凝血盒等位基因交换突变在衣原体沙眼

在血清型 1 型肺炎链球菌 菌株 519/43 中构建突变体

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

的产生

在型号蓝藻种类标记和无标记突变体的产生

在血清型 1 型肺炎链球菌菌株 519/43 中构建突变体