我感谢乔治教会实验室 pJA1 质粒的那种礼物。我感谢法比耶纳汉堡和亚历克斯 Boehm 从 Jenal 实验室在 Biozentrum 在巴塞尔的帮助与细菌共轭和提供 BW20767 菌株。我也感谢奥林 Silander 的帮助编辑。这项研究的经费来自于新西兰梅西大学的基金和瑞士巴塞尔大学的系统生物学 ("战斗 X" 项目 "Bumann") 的瑞士倡议。

警告: 如果使用 . 福氏 作为接收方应变, 请注意, s. 福氏 是一种可以引起胃肠道疾病的人病原体。涉及 福氏 的实验必须使用适当的生物安全预防措施 (在欧洲和美国指定的 BSL-2 或新西兰的 PC2) 进行。在与该协议相关的视频中执行的所有实验都是使用 PC1 设置中的致病 大肠杆菌 的良好受体应变 MG1655 进行的。与 志贺菌福氏 一起工作以前是在巴塞尔的 Biozentrum 的 BSL-2 实验室中执行的, 如 6 中所述。
 注意: 下面的实验有效地完成了两次。第一部分 (步骤 1.1-步骤 4.5) 是为了找到最好的稀释镀出的图书馆, 其中的目标是找到一个稀释的共轭, 这给了许多单独的殖民地每个板块, 但没有这么多的草坪形式。这是为了减少突变体之间的竞争, 显著降低体质和那些更强健的健身。第二部分 (步骤 5.1-7.4) 是最后的图书馆的创作, 许多板材是传播的适当的稀释图书馆. 
 1. 在试验前一天准备 <ol> <li> 从冷冻库存接种, 2 毫升的 LB 肉汤中的供体菌株, 米勒食谱 (氯化钠10克/升) 培养基中含有氨苄西林的 100-#181; 克/毫升。这里使用的施主菌株是 大肠杆菌 菌株 BW20767 15 窝藏 pJA1 质粒 11 。使用氨苄西林保持选择 pJA1 质粒. </li>
	<li> 接种, 从单一的殖民地或冷冻的股票, 受体菌株在2毫升的 LB 介质与抗性标记除氨苄青霉素或卡那霉素。这里使用的接收株是 大肠杆菌 和 #34; 野生类型和 #34; 应变 MG1655 17 , 18 , 带有自发电阻突变体的啶酸。它生长在30和 #181; g/毫升啶酸. </li>
	<li> 准备 20 Luria 汤含有1.5% 琼脂板 (在 90 mM 培养皿中, 约12.5 毫升)。缺乏抗生素的琼脂板可以储存在4和 #176; C 在使用前几个月. </li>
	<li> 准备 20 Luria 汤, 含有1.5% 琼脂板 (在90毫米的培养皿中, 约12.5 毫升) 含有啶酸在30和 #181; 在50和 #181 中的 g/毫升和卡那霉素; g/毫升 (LBA Nal30 Kan50)。含有抗生素的琼脂板可以在4和 #176 的黑暗中储存, 直到使用数月. </li>
	<li> 准备200毫升的无菌 LB 介质。LB 介质可在室温下储存数月. </li>
	根据制造商和 #39 的说明, <li> 准备1毫升的 IPTG 100 mM 无菌库存. </li>
</ol> 2。细菌交配或共轭 <ol> <li> 在室温下在 1.4万 x g 上离心1毫升的供体应变过夜培养1分钟。放弃增长媒体。采取这一步骤是为了消除所有含有抗生素的培养基. </li>
	<li> 重将细胞小球转化为110和 #181; l 的 LB (不含抗生素), 从而集中的文化. </li>
	<li> 使用无菌钳, 放置一个无菌的硝化纤维素过滤器 (或者是一个无菌的印迹纸可以使用, 见材料列表) 到一个 LBA 板块中间 (不含抗生素)。给盘子和 #34 贴上标签; 负控制: 施主和 #34;. </li>
	<li> 使用吸管, 下降50和 #181; L 的集中捐赠文化到过滤器上. </li>
	<li> 对接收方应变重复步骤 2.1-4, 标记板和 #34; 负控制: 收件人. #34; </li>
	<li> 对于库, 请将50和 #181; l 的施主 (从步骤 2.2) 和50和 #181; l 的受体应变 (从步骤 2.5) 到一个单一的无菌过滤器上的 LBA 板 (这些板块不应该含有抗生素)。给这个盘子和 #34 贴上标签; 库. 和 #34; 交配发生是因为捐赠者和接收者在过滤器上有密切的物理接触. </li>
	<li> 将含有过滤器的琼脂板放置在37和 #176; C 用于 6 h. </li>
</ol> 3。pJA1 转的活化 IPTG 转 <ol> <li> 准备三15毫升的圆锥小瓶, 其中含有2毫升的 LB 与 IPTG 在最后浓度1毫米 ( 即 , 20 和 #181; L 100 毫米库存 IPTG)。标签: 捐助者控制、收件人控制和库. </li>
	<li> 在37和 #176 增长 6 h 后从孵化箱中取出板; C (从步骤 2.4)。在过滤器上应该可以看到一些细菌生长. </li>
	<li> 使用无菌钳, 取出供者控制的滤纸, 并将其放置在15毫升圆锥小瓶中, 标记为供者控制 (从步骤 3.1)。对收件人控件和库执行相同的方法. </li>
	<li> 点击管子将滤纸放到15毫升圆锥小瓶的底部, 确保它完全浸没在 LB 中. </li>
	<li> 涡流管至少有一分钟的高, 以解除细菌从硝化纤维素过滤器。LB 应该成为多云与细菌细胞 ( 图 2 ). </li>
	<li> 使用无菌玻璃微珠或无菌吊具, 板200和 #181; vortexed 细菌悬浮从施主控制到 LBA Nal30 Kan50 板标记和 #34;D onor 控制, 200 和 #181; l 和 #34;. </li>
	<li> 对收件人控件重复以上步骤 (3.6), 标记板和 #34; 收件人控件、200和 #181; L 和 #34;... </li>
	<li> 重复上述步骤 (3.7), 为库, 标签的板和 #34; 图书馆, 200 和 #181; 我和 #34... </li>
	<li> 稀释 ( 即 、1:5、1:10 和1:100 稀释, 使用 LB 不含抗生素作为稀释剂) 进行库。板200和 #181; 未经稀释的图书馆和额外的稀释到适当标记 LBA Nal30 Kan50 板. </li>
	<li> 将板放入37和 #176; C 孵化器 18 h. 克隆与转插入的基因, 导致大量的健康损失可能需要更长的时间生长和出现在板块上。应该有多种菌落大小 ( 图 3 )。捐献者和受体的应变片不应该有殖民地. </li>
</ol> 4。选择要用于最终的变种库的库的适当稀释 <ol> <li> 确定从步骤3.9 生成的库的稀释量, 它会产生许多大小不等的菌落, 它们之间的间隔是适当的。其目的是在一个板块上获得尽可能多的殖民地, 但没有那么多殖民地相互融合并争夺资源。这个数字应该是大约 500-2, 000 殖民地每个板材。在 图 3 中显示了一个在平板上适当的菌落密度的示例. </li>
	<li> 记录车牌上的菌落数. </li>
	<li> 计算共轭频率 (每个收件人单元格的 conjugants 数)。此范围应为 1 x 10 -4 到 1 x 10 -6 19 。
	<li> 根据下游应用程序确定最终库中需要的变种人的数量。许多用户只需要一个尽可能多的变种人的库。要产生尽可能多的转插入, 理论上是可能的 (一个插入每个基对), 目的是近似与基因组中的基底对相同数量的菌落数 ( 即 , 4.5 x 10 6 用于 大肠杆菌 ), 以完全饱和的基因组与所有可能的转插入。重要的是要注意的是, 许多变种人在关键或功能重要的基因中的插入, 将无法恢复, 因为这些突变将是致命的收件人. </li>
	<li> 计算创建所需大小的库所需的初始库卷的数量。例如, 所需的库大小为 5 x 10 4 变种。从步骤3.9 的殖民地的最佳间距的稀释是200和 #181; 1:10 稀释的 L。在这个板块上的菌落数量估计大约是2000殖民地。如果需要 5 x 10 4 变种, 并且每个板块都有2000个菌落, 那么在这种稀释的时候需要25板块. </li>
</ol> 5。创建最终的变种库 <ol> <li> 重复步骤1到 3.8, 根据需要调整步骤1.4 中的板数 (请参见步骤 4.4). </li>
	<li> 在步骤3.9 中, 将步骤4.1 中选择的稀释板放到所需的尽可能多的板上, 以创建所需大小的库 (请参见步骤 4.4). </li>
</ol> 6。估计库密度 <ol> <li> 计算每个板块有多少个菌落, 从而粗略估计出图书馆有多稠密。例如: 共有 2x10 5 殖民地被镀了。 大肠杆菌 MG1655 基因组大约为 4.5 x 10 6 基对。因此, 一个具有大约 2 x 10 5 插入的库意味着每个22基对都有一个插入 平均 , 并且每个基因应该被突变约45次, 因为一个基因大约是 1 x 10 3 基对。在这个库中, 基本基因的插入很可能是很低的, 因此非必要基因的密度可能会比这更大。这类计算提供了一个转密度的广泛估计. </li>
</ol> 7。池转库和存储 <ol> <li> 计数完菌落后, 将1毫升 (或更多) 的 LB (必要时) 添加到库板上, 并使用无菌的吊具将细菌从盘子中刮掉。去除细菌悬浮和放置在一个50毫升或15毫升圆锥小瓶。重复所有的盘子. </li>
	<li> 漩涡池中的细菌悬浮了整整一分钟来质悬浮. </li>
	<li> 将无菌甘油添加到最终浓度为 20%. 
	<ol> <li> 准备 100 x 20 和 #181; L 等分在0.25 毫升的管子中, 以方便再生. </li>
		<li> 在 cryovials 中至少准备两个或多个1毫升分. </li>
	</ol> </li> <li> 将所有等分存储在-80 和 #176; C </li> </ol>

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</ol>

作者没有什么要透露的。

这里描述的协议允许构造一个密集的插入库。此方法允许使用 5 mL 的区域性卷6创建具有超过 2 x 105唯一转变种的转库。这是相对容易执行, 使用试剂在大多数基本的微生物学实验室, 是可伸缩的, 并要求在昂贵的设备或消耗品, 如电穿孔试管的方式很少。
该方法的一个显著优点是, 在理论上, 用户在选择 enterobacterial 受体菌株时具有很大的自由度。本文以及其他11, 使用大肠杆菌作为接收菌株, 但 pJA1 质粒已成功地用于其他 enterobacterial 受体的物种, 如志贺菌福氏 6和沙门氏菌 enterica螺旋体鼠伤寒杆菌 SL134410。理论上, 在 pJA1 中复制的γ原点 (oriR6Kγ) 允许此质粒在广泛的主机范围19中进行维护, 从而使收件人应变为红外 +.最近, 已经描述了新的方法, 允许在一系列的 enterobacterial 菌株20中构造红外 + , 从而提供了额外的灵活性。此外, 在 pJA1 的 RP4 质粒中的300 基对暴民区域允许共轭将此质粒转移到广泛的革兰阴性菌菌株19。简单地说, 这种方法理论上可以用于各种受体菌株, 只要满足几个条件: 该菌株是红外+, 并标明了除卡那霉素以外的抗生素抵抗, 除了供体菌株。
协议中的一个关键步骤是在步骤4.1 中估计出适当数量的单元格。如果蜂群间隔太近, 它们就会在板块上争夺养分, 而不适合的突变体则超过。这可能导致突变体总数的减少。另外, 如果殖民地间隔太远, 将有太少的殖民地在板块上, 和总数量的琼脂板需要实现一个大型图书馆变得繁重。因此, 以每个板块的菌落数来实现正确的平衡是很重要的。
执行协议中列出的控件非常重要, 以确保步骤按所述的方法工作。值得注意的是, 当使用啶酸作为 counter-selection 对供体菌株, 重要的是要确保负施主控制板是没有殖民地。这是因为可能有一个低速率 (大约 1 x 10-10) 的21自发抗啶酸, 产生假阳性。通常, 共轭和移位的速率大约是 2 x 10-4 19。因此, 共轭和转位率是几个数量级大于自发抗啶酸的速率。因此, 与真实换位事件相比, 误报率非常低, 在协议工作时被视为微不足道。然而, 如果共轭或转位率明显降低, (从低交配效率和/或缺乏诱导的转基因与 IPTG) 和协议的规模, 以弥补这一点, 然后假阳性的数量 (克隆没有插入的转) 也可能增加。
在步骤3.2 和3.10 中, 可以对孵化时间进行一些修改。步骤3.2 指出, 共轭应该发生在6小时, 但在我们的经验中, 这个时间步长可以变化 (即, 4-7 h), 而不会显著改变结果。此外, 在步骤3.10 中, 还可以调整菌落在琼脂板上孵育的时间长度。这可以根据接收菌株的平均加倍时间或生长速率而变化。此外, 在我们的经验中, 18 h 产生了不同的菌落大小, 表明一个不同的健身库。然而, 具有极大降低体质的菌落可能需要更长的时间才能生长, 因此在18小时后可能不可见。如果这种方法被用来寻找极少的适应性克隆, 更长的潜伏期和较少的殖民地在板块上减少拥挤 (即, 48 ĥ, 50-300 殖民地) 可以使用。
这种方法的附加缺陷包括: 不能使用已经卡那霉素抵抗的受体菌株。pJA1 质粒中的卡那霉素抗性标记物可以替代可选的标记物, 如氯霉素耐药性以克服这一障碍。同样值得注意的是, 理论上, 有可能使用一种抗氨苄西林的受体菌株, 因为含有氨苄西林耐药标记的 pJA1 质粒在换位后不久就会丢失。
在选择的遗传背景下建立一个稠密的转库, 对于许多下游应用来说都是潜在的有利条件。例如, 一个稠密的转库可以用来识别缺陷突变体使用副本电镀22或确定在建立感染的变种人是有缺陷的1,2。最近, 随着 dna 测序成本的下降和下一代测序技术等新技术的普及, 转图书馆已被用于深入 dna 测序, 以深入了解基因的重要性、基因功能和遗传相互.其中一些方法在23中进行了审阅, 其中包括转定向插入站点测序 (TraDIS)、转测序 (Tn-seq)、高吞吐量插入跟踪 (按深度测序 (命中)) 和插入顺序 (INSeq)。所有这些下游方法都依赖于密集的转插入库的构造。虽然其他向量可能需要用于特定的下游方法, 但此处描述的协议概述了要遵循的显著程序点。

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56216/creation-dense-transposon-insertion-library-using-bacterial">Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri</a>. The Discussion and References sections have been corrected.
The second paragraph in the Discussion section was updated from:
A significant benefit of this method is that, in theory, the user has wide latitude in the choice of enterobacterial recipient strains. This paper, as well as others11, use&#160;E. coli&#160;as a recipient strain, however the pJA1 plasmid has been used successfully with other enterobacterial recipient species such as&#160;Shigella flexneri6&#160;and&#160;Salmonella enterica&#160;serovar Typhimurium strain SL134410. Theoretically, the &#947; origin of replication (oriR6K&#947;) in pJA1 allows this plasmid to be maintained in a broad host range19, allowing that the recipient strain is&#160;pir+.&#160;Recently, new methods have been described that allow for construction of the&#160;pir+&#160;in a range of enterobacterial strains20, giving additional flexibility. Additionally, the300 base pair mob&#160;region from the RP4 plasmid in pJA1 allows conjugative transfer of this plasmid to a wide range of gram negative bacterial strains19. Simply put, this method could theoretically be used with a variety of recipient strains, as long as several conditions are met: the strain is&#160;pir+, and is marked with an antibiotic resistance other than kanamycin and other than the donor strain.
to:
A significant benefit of this method is that, in theory, the user has wide latitude in the choice of enterobacterial recipient strains. This paper, as well as others11, use&#160;E. coli&#160;as a recipient strain, however the pJA1 plasmid has been used successfully with other enterobacterial recipient species such as&#160;Shigella flexneri6&#160;and&#160;Salmonella enterica&#160;serovar Typhimurium strain SL134410. Theoretically, the &#947; origin of replication (oriR6K&#947;) in pJA1 allows this plasmid to be maintained in a broad host range19, allowing that the recipient strain is&#160;pir-.&#160;The&#160;300 base pair mob&#160;region from the RP4 plasmid in pJA1 allows conjugative transfer of this plasmid to a wide range of gram negative bacterial strains19. Simply put, this method could theoretically be used with a variety of recipient strains, as long as several conditions are met: the strain is&#160;pir-, and is marked with an antibiotic resistance other than kanamycin and other than the donor strain.
The References&#160;section was updated from:
<ol start="18">
	<li>Blattner, F. R., Plunkett, G.,&#160;et al.&#160;The Complete Genome Sequence of Escherichia coli K-12.&#160;Science.&#160;277&#160;(5331), 1453-1462 (1997).</li>
	<li>Alexeyev, M. F., &#38; Shokolenko, I. N. Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of gram-negative bacteria.&#160;Gene.&#160;160&#160;(1), 59-62 (1995).</li>
	<li>Kvitko, B. H., Bruckbauer, S.,&#160;et al.&#160;A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids.&#160;BMC Res Notes.&#160;5&#160;(1), 157 (2012).</li>
	<li>Barrick, J. E., Yu, D. S.,&#160;et al.&#160;Genome evolution and adaptation in a long-term experiment with Escherichia coli.&#160;Nature.&#160;461&#160;(7268), 1243-1247 (2009).</li>
	<li>Jacobs, M. A., Alwood, A.,&#160;et al.&#160;Comprehensive transposon mutant library of Pseudomonas aeruginosa.&#160;PNAS.&#160;100&#160;(24), 14339-14344 (2003).</li>
	<li>van Opijnen, T., &#38; Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.&#160;Nature Rev Microbiol.&#160;11&#160;(7), 435-442 (2013).</li>
</ol>
to:
<ol start="18">
	<li>Blattner, F. R., Plunkett, G.,&#160;et al.&#160;The Complete Genome Sequence of Escherichia coli K-12.&#160;Science.&#160;277&#160;(5331), 1453-1462 (1997).</li>
	<li>Alexeyev, M. F., &#38; Shokolenko, I. N. Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of gram-negative bacteria.&#160;Gene.&#160;160&#160;(1), 59-62 (1995).</li>
	<li>Barrick, J. E., Yu, D. S.,&#160;et al.&#160;Genome evolution and adaptation in a long-term experiment with Escherichia coli.&#160;Nature.&#160;461&#160;(7268), 1243-1247 (2009).</li>
	<li>Jacobs, M. A., Alwood, A.,&#160;et al.&#160;Comprehensive transposon mutant library of Pseudomonas aeruginosa.&#160;PNAS.&#160;100&#160;(24), 14339-14344 (2003).</li>
	<li>van Opijnen, T., &#38; Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.&#160;Nature Rev Microbiol.&#160;11&#160;(7), 435-442 (2013).</li>
</ol>

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56216/creation-dense-transposon-insertion-library-using-bacterial">Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri</a>.&#160;The Discussion and References sections have been corrected.

Erratum: Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

细菌中转诱变文库的建立对各种应用都是有用的, 从细菌病原体的毒力基因的发现到1,2, 到基本基因的研究3,4,5,6, 到遗传交互网络的标识7,8,9。这些研究的关键是能够创建一个大的突变体库。使用转 (随机插入基因组的 DNA 的短片段) 是扰乱基因功能的简单手段, 因为在一个基因的开放阅读框架或调控区域内插入一个转, 通常会扰乱功能或表达的基因。
本文概述了使用质粒 pJA110在大肠杆菌或S. 福氏中创建转库的方法。使用这种质粒有两个主要优点。第一个好处是, Tn10 转表达的变体 pJA1 质粒是诱导和低插入偏置11,12, 意味着转将随机集成到基因组时异丙酯β-d--1-thiogalactopyranoside (IPTG) 被添加到介质中。转含有卡那霉素电阻标记, 允许选择突变体与转插入到受体菌株的染色体。pJA1 质粒的第二个优点是它包含了一个 R6K 突变的复制源。复制的 R6K 突变源需要 lambda红外基因来维护质粒的13。由于质粒不能在红外-菌株中复制, 因此它将在接收株中丢失 (图 1)。这确保了 Tn10 转从细胞中移除, 不再活动, 减少了在初始换位事件之后的进一步突变。
利用细菌结合将 pJA1 质粒从供体菌株转移到受体菌株是有利的, 原因有几个。共轭是简单和廉价的执行, 不需要特殊设备, 如 electroporator。此外, 细菌结合的高效率, 使一个非常大的图书馆 (和 #62; 2 x 105独特的插入) 要达到仅几毫升 (毫升) 夜间细菌培养6。该过程需要大约两个小时的动手时间, 随着时间的孵化和成长的细菌。Langridge et al.14报告了执行 130 electroporations 以创建一个类似于此处所述8的大小的转诱变库, 它是通过单个共轭来实现的。使用 130 electroporations 需要劳力密集和费时的准备 electrocompetent 细胞和使用许多昂贵的材料 (如如, 电穿孔试管), 成本超过1000美元的消耗品单。其他研究10使用了类似的方法, 但不同的细菌菌株, 并取得了比这里报道的更小的库大小 (5 x 104菌落形成单位)。
关于施主菌株的注: 这里使用的供体菌株是大肠杆菌菌株 BW2076715包含 pJA1 转质粒16。应变 BW20767 可以共轭到其他菌株, 使 pJA1 质粒的转移高效。此外, 重要的是, BW20767 是 lambda红外 +.如前所述, 质粒 pJA1 仅能在含有 lambda红外基因的菌株中维持。这种菌株是卡那霉素和氨苄西林耐药性。pJA1 质粒含有氨苄西林抗性标记, 转中含有卡那霉素抵抗标志物。在100µg/毫升使用氨苄西林的质粒上选择这种菌株生长。还值得注意的是, 其他菌株, 窝藏组成表达的转基因已知是不稳定的, 而转这里使用的是在诱导控制下, 仍然有低的风险, 泄漏的表达。因此, 建议将这一菌株的通过减至最低, 并从冰冻文化中为每一个新的图书馆准备工作, 采取新的条纹。这里使用的供体菌株可在我们的实验室索取。
关于受体菌株的说明: 受体菌株可能是一种选择, 如 K12-derived 实验室菌株的大肠杆菌或菌株的S. 福氏(也请参阅讨论).受体菌株必须有一个额外的抗生素抗性标记, 不是卡那霉素的阻力, 因此, 供体菌株可以选择反对。对于受体菌株, 一个大肠杆菌MG1655 菌株在这里使用引入自发啶酸突变。自发啶酸突变体的选择是在200µL 等分上镀出2毫升的过夜培养物, 在含有啶酸的30µg/毫升的板上。选择一个单一的克隆 MG1655 是抗啶酸成为受体菌株。此外, 如上所述, 收件人应变必须是 lambda红外负。
概述: 一旦细菌结合发生, pJA1 质粒已从施主菌株转移到受体菌株, IPTG 向培养基的添加将导致tnp基因的表达, 这是在 IPTG 诱导的控制下lacIq/Ptac 启动子 (图 1)。pJA1 上的tnp基因是一个突变转, 它在热点处的插入频率较低,6,10,11。在加入和诱导与 IPTG, 转被激活和随机插入的基因组。质粒不能在λ受体菌株中维持, 并且丢失。

<table><tbody><tr><td>0.45 micron nitrocellulose filters (sterile)</td><td>Millipore</td><td>HAWP02500</td><td>Alternatively blotting paper can be used (listed below)</td></tr><tr><td>Blotting paper cut into ~ 1 inch circles, then autoclave/sterilized in foil. There is no difference in efficiency between the nitrocellulose filters or blotting paper</td><td>GE life Sciences: product Grade 3MM Chr Blotting Paper, sheet, 46 &amp;times; 57 cm</td><td>3030-917</td><td>Alternatively nitrocellose filters can be used (listed above)</td></tr><tr><td>Metal Forceps, sterile</td><td>VWR/Global Science New Zealand</td><td>MURRE009/01</td><td></td></tr><tr><td>Petri dishes, sterile, 90 mm diameter, 68 mL volume,&amp;nbsp; 12.5 mL working volume.</td><td>Interlab New Zealand</td><td>2303-1090</td><td></td></tr><tr><td>Sterile glass spreaders</td><td>VWR/Global Science New Zealand</td><td>NZNZSPREADER</td><td>Alternatively sterile glass beads can be used for spreading culture onto plates (listed below)</td></tr><tr><td>Sterile glass beads</td><td>VWR/Global Science New Zealand</td><td>HERE1080603</td><td>Alternatively a sterile glass spreader can be used for spreading culture onto plates (listed above)</td></tr><tr><td>Nalidixic acid (optional)</td><td>GoldBio.com</td><td>N-015-250</td><td></td></tr><tr><td>Ampicillin sodium salt BioChemica</td><td>VWR/Global Science New Zealand</td><td>APLIA0839.0025</td><td>prepare according to manufacturer recommendation</td></tr><tr><td>Kanamycin sulfate BioChemica</td><td>VWR/Global Science New Zealand</td><td>APLIA1493.0010</td><td></td></tr><tr><td>IPTG (Isopropyl &amp;beta;-D-1-thiogalactopyranoside)&amp;nbsp;</td><td>Thermofisher New Zealand</td><td>FMTR0393</td><td></td></tr><tr><td>Difco Agar granulated</td><td>Fort Richard Laboratries/Interlab New Zealand</td><td>214530</td><td></td></tr><tr><td>Luria Broth Base (Miller&#039;s LB Broth Base)</td><td>Thermofisher</td><td>12795-084</td><td></td></tr><tr><td>Glycerol bidistilled 99,5 %</td><td>VWR/Global Science New Zealand</td><td>VWRC24388.320</td><td></td></tr><tr><td>15 ml polypropelene sterile conical vials&amp;nbsp;</td><td>VWR/Global Science New Zealand</td><td>CORN430791</td><td></td></tr><tr><td>Microcentrifuge tubes, flat cap, 1.7ml, Ultra-clear PP, graduated, autoclavable and freezable</td><td>VWR/Global Science New Zealand</td><td>AXYGMCT-175-C</td><td></td></tr><tr><td>50ml Centrifuge tubes, Falcon, PP, conical, printed graduation, with flat screw caps, sterile</td><td>VWR/Global Science New Zealand</td><td>BDAA352070</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Incubator at 37C</td><td></td><td></td><td></td></tr><tr><td>Autoclave for sterilizing media&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

creation of a dense transposon insertion library using bacterial conjugation in enterobacterial strains such as escherichia coli or shigella flexneri

转突变是一种方法, 通过随机基因组插入一个叫做转的 DNA 片段, 允许转基因。下面的协议概述了一种在含有卡那霉素抵抗标记的转质粒的细菌菌株间高效转移的方法。质粒载体的转是由一个变体的tnp基因编码的, 它将转插入到受体株的基因组中, 插入偏低。因此, 这种方法允许创建大的突变库, 其中转已插入到独特的基因组位置的受体菌株的大肠杆菌或志贺菌福氏细菌。通过使用细菌结合, 而不是其他方法, 如电穿孔或化学转化, 大型图书馆与数十万独特的克隆可以创建。这会产生高密度的插入库, 插入的频率与非必要基因中的每4-6 碱基对一样频繁。此方法优于其他方法, 因为它允许使用廉价、易用和高效的方法创建密集的转插入库。转库可用于下游应用, 如转测序 (Tn-Seq), 以推断基因相互作用网络, 或更简单地说, 在突变 (前瞻性遗传) 屏幕。

经过18小时的增长, 板块应该包含许多殖民地大小的各种殖民地的 ( 图 3)。不同的菌落大小指示不同适应性的克隆, 这是该协议行之有效的好兆头。捐赠者和受赠人的控制在板块上应该没有增长。使用上述协议应能产生超过 2 x 105的菌落形成单元 (CFU)。将协议扩展到五复制动词一次应生成大约 1 x 106 CFU。在以前的工作中, 使用下一代测序的分析表明, 这样一个规模扩大的图书馆应该产生和 #62; 2 x 105唯一插入6。在非常高的 CFU (即, 1 x 106 CFU) 中, 唯一插入的速率预计会趋于平稳, 因为所有可用的 (非致命) 插入都将被表示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56216/56216fig1.jpg"> 
图 1: 细菌结合和转插入染色体的示意图.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56216/56216fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56216/56216fig2.jpg"> 
图 2: 滤镜的图像在涡流之前和之后的 LB 介质中水.在涡流之前, 细胞被粘在过滤器上, 介质是清晰的。涡流后, 当细胞从过滤器中脱落并在介质中时, 介质变得浑浊。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56216/56216fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56216/56216fig3.jpg"> 
图 3: (A) 转库的代表性板块经过18小时的生长。(B) 关闭菌落大小。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56216/56216fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri at JoVE.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

这里介绍的是一个简单的方法, 创建高密度转插入库在大肠杆菌或志贺菌福氏使用细菌共轭。该协议允许通过随机的基因组插入转在细菌中创造出数十万个独特的突变体。

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

creation-dense-transposon-insertion-library-using-bacterial

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

15.9K 次观看.  Massey University. 这里介绍的是一个简单的方法, 创建高密度转插入库在大肠杆菌或志贺菌福氏使用细菌共轭。该协议允许通过随机的基因组插入转在细菌中创造出数十万个独特的突变体。

视频: Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

科研

遗传学

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

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 ...

生物工程

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

用 BIBAC 二元矢量生成单拷贝插入的转基因植物

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

细菌结合频率的高分辨率比较

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

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 ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

免疫与感染

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 ...

生物学

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

在革兰氏负性细菌中生成跨波子插入库，用于高通量测序

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify&#160;molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts.
Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is&#160;carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

This is an adapted method for identifying candidate insect colonization factors in a Burkholderia beneficial symbiont. The beetle host is infected with a random mutant library generated via transposon mutagenesis, and library complexity after colonization is compared to a control grown in vitro.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

摘要

探索更多视频

接合交配测定在接合介入转移蛋白的序列特异性分析

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

网站特定的细菌染色体工程:ΦC31整合介导的盒式交换（IMCE）

用 BIBAC 二元矢量生成单拷贝插入的转基因植物

细菌结合频率的高分辨率比较

大肠杆菌中天然偶联质粒介导的水平基因转移检测

高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性

的产生

在革兰氏负性细菌中生成跨波子插入库，用于高通量测序

跨波森插入测序作为工具，以阐明细菌殖民因素在拉克霍尔德里亚角膜甲虫

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

摘要

探索更多视频

接合交配测定在接合介入转移蛋白的序列特异性分析

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

网站特定的细菌染色体工程:ΦC​​31整合介导的盒式交换（IMCE）

用 BIBAC 二元矢量生成单拷贝插入的转基因植物

细菌结合频率的高分辨率比较

大肠杆菌中天然偶联质粒介导的水平基因转移检测

高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性

的产生

在革兰氏负性细菌中生成跨波子插入库，用于高通量测序

跨波森插入测序作为工具，以阐明细菌殖民因素在拉克霍尔德里亚角膜甲虫

网站特定的细菌染色体工程:ΦC31整合介导的盒式交换（IMCE）