我们要感谢 MGH 崔德威尔虚拟图书馆的丽莎 Philpotts 她在数据库搜索中的指导。这项工作得到了囊性纤维化基金会 (YONKER16G0 和 HURLEY16G0) 和 NIH NIAID (BPH 和艾德: R01 A1095338) 的支持。

作者报告没有任何金融利益冲突。埃利安娜 Drenkard 和弗雷德里克奥苏贝尔参与创建 PA14 非冗余座子突变库。布赖恩赫尔利和 Lael Yonker 目前的房子和分发的变种人图书馆作为一部分的儿科在马萨诸塞州总医院。

P。铜绿假单胞PA14NR 集是科学界的宝贵资源。根据2017年3月数据集从 Clarivate 分析的基本科学指标数据库, Liberati et 等。(2006)37描述了 PA14NR 集的构造, 排名在微生物学刊物的前1% 位。谷歌学者报告了超过 600 Liberati 的引文et 。(2006) 2017年8月原稿。图书馆在阐明铜绿假单胞菌发病机制方面发挥了重要作用。重要的是, PA14NR 集的96井板格式方便了用于研?...

铜绿假单胞菌是一种表型和 genotypically 不同和适应性的革兰阴性菌, 存在于土壤、水和大多数人类环境中, 以及皮肤微生物区系。与许多细菌种类相比, 铜绿假单胞菌有一个相对较大的基因组 5.5-7 的 Mbp 与高 G + C 含量 (65-67%)。此外, 它的相当一部分基因参与代谢适应性和管理网络的一部分, 允许极大的灵活性, 以应对环境压力1。铜绿假单胞菌表达了过多的毒力因素, 表现出形成生物膜的倾向, 具有通过多种仲裁传感通路协调反应的能力, 并显示出开发耐药性的显著能力。和公差2,3,4,5,6,7,8。这些属性对治疗由铜绿假单胞菌引起的感染带来了重大挑战。
慢性的铜绿假单胞菌感染可能发生在许多疾病状态。囊性纤维化 (CF), 一种遗传性疾病引起的突变的囊肿纤维化跨膜电导调节器 (CFTR)基因, 导致浓缩, 感染的分泌物在呼吸道, 渐进性支气管扩张, 最终, 死亡从呼吸衰竭9。成年后, CF 的大多数患者慢性感染铜绿假单胞菌, 这在发病率和死亡率与此疾病相关的10中起着关键作用。此外, 严重烧伤损伤的患者11、tracheostomies12、联合替换13或留置导管14有风险的P. 铜绿假单胞菌感染与细菌形成的能力有关生物膜和逃逸宿主炎症响应15。此外, 在通过广谱、连续抗菌治疗选择了多抗生素耐药性或耐受性的人群后, 在无竞争的情况下进行殖民化,12,16,17,18. 更好地了解铜绿假单胞病毒的发病机制将对许多疾病状态产生重大影响。
大量研究了几种铜绿假单胞菌临床分离株, 包括 PAO1、PA103、PA14 和白脓杆菌等, 探讨了铜绿假单胞病毒发病机制的不同特点。应变 PA14 是一种临床隔离, 属于世界上最常见的克隆组之一, 在全球范围内19,20 , 并没有在实验室广泛传代。PA14is 在脊椎动物感染模型中具有剧毒, 具有显著的内毒素特征21, 毛结构22, 致病性群岛23, III. 型分泌系统 (TTSS), 对哺乳动物细胞的细胞毒性24和抗生素耐药性和持久性的配置文件25。此外, PA14 在众多寄主病原体模型系统中也具有剧毒, 包括植物叶片浸润模型26,27,秀丽线虫感染模型28, 29, 昆虫模型30,31, 以及老鼠肺炎模型32,33和皮肤烧伤模型34。
基因组范围内的突变体库是不必要基因中的等基因系突变体的集合, 它构成了一种非常有力的工具, 可以通过基因组尺度分析来了解生物体的生物学。目前, 在铜绿假单胞菌中构建的两个近饱和座子插入突变库可供分发。已为两个库确定了随着转座子的插入位置。这些所谓的非冗余图书馆促进了对细菌株的全基因组研究, 大大减少了筛选 uncharacterized 随机座子突变体所涉及的时间和成本。由华盛顿大学随着转座子实验室策划的 PAO1 座子突变库, 在 PAO1 使用 phoA 的 MPAO1 隔离中构建, 是lacZ/哈,是Manoil/哈35。该图书馆由序列验证的收集9437座子突变体提供广泛的基因组覆盖, 包括两个突变体的大多数基因36。有关铜绿假单胞菌PAO1 座子突变库的信息, 可在 http://www.gs.washington.edu/labs/manoil/libraryindex.htm 的公共、互联网访问的 Manoil 实验室网站上查阅。PA14 应变非冗余座子插入突变库 (PA14NR 集) 构造的应变 PA14 使用随着转座子MAR2xT7 和TnphoA 37 目前由儿科部分发在马萨诸塞州总医院。PA14NR 集包括5800多个变种人的集合, 在非必需基因37中单座子插入。有关 PA14NR 集建设的详细信息, 在公开、互联网访问的网站 http://pa14.mgh.harvard.edu/cgi-bin/pa14/home.cgi？section=NR_LIB 中介绍, 其中还包含各种在线搜索工具, 以便利使用 PA14NR设置。
原始的 PA14NR 集合包括5459个变种人, 从一个综合图书馆选择大约3.4万个随机座子插入突变体, 对应于4596预测 PA14 基因代表77% 的所有预测 PA14 基因37。自从2006年图书馆的建设新变种增加, 目前 PA14NR 集包括超过5800变种人38 , 代表大约 4600 PA14 基因。大多数 PA14 座子突变体都是在野生类型背景37中生成的。有关变种人库的每个成员的详细信息, 包括遗传背景, 可通过搜索联机数据库或下载非冗余库电子表格 (PA14 网站上提供的两种功能) 提供 (http://pa14. mgh 哈佛大学/cgi-bin/pa14/主页. cgi)。大多数变种人是使用 MAR2xT7 (MrT7) 座子创建的, 使用 TnPhoA (phoA) 座子37创建的小集。每个座子有一个抗生素耐药性盒, 它允许突变选择使用庆大霉素 (MrT7) 或卡那霉素 (phoA)。PA14NR 组的突变体存储在六十三96井板中, 包括两个额外的96井控制板, 由野生型 PA14 接种和适合井插在预设模式下组成。与在线搜索工具配对的96井板格式极大地促进了筛选测试的定制开发, 使用户能够轻松地识别与突变体表型相关的基因。在线搜索工具还有助于搜索和选择进一步研究所需的其他相关突变体。
PA14 和 PAO1 座子突变体库是科学界非常重要的全球资源, 它们相互补充, 验证了该病原菌的未知基因和通路的功能。巧合的是, 自从 PAO1 和 PA14 座子突变库的构建以来, 许多铜绿假单胞菌的全基因组 DNA 测序分析表明 PAO1 和 PA14 属于不同的主要 subclades 的铜绿假单胞菌系统系统7,39,40,41。由于临床上的铜绿假单胞菌分离物分布在整个系统发育过程中, PAO1 和 PA14 属于不同的铜绿假单胞子组的事实提高了两个座子变异库的价值, 以便比较研究。
关于细菌突变库的构建和筛选的出版物, 包括铜绿假单胞库35,37,42, 在文献中很容易获得。但是, 根据我们的知识, 没有发布的协议描述用于复制、维护和验证细菌突变库的详细程序和技术。
本出版物中概述的方法描述了一套三协议, 便于使用和维护 PA14NR 集。第一个协议描述了向 PA14NR 集的收件人推荐的库的复制。第二个协议包括用于裸奔、生长和存储使用 PA14NR 集识别的单个突变体的准则。第三个协议描述质量控制技术, 包括 PCR 扩增的片段从座子突变体和后续测序, 以确认变种人的身份。这组协议也可用于复制和维护其他细菌突变库或集合。强烈建议复制细菌突变库或集合, 以保持 "主副本" 的完整性 (收到的原始副本)。复制 PA14NR 集的几个副本用于常规实验室使用, 可以最大限度地减少主拷贝井间踪污染的可能性。

<table>
	<tbody>
		<tr>
			<td>Materials for Library Replication</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 96-well Tissue-culture treated, case of 50</td>
			<td>Corning Life Sciences</td>
			<td>353072</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Sterile 96 Well Clear V-Bottom 2000&#956;L Deep Well Plates, case of 25</td>
			<td>Corning Life Sciences</td>
			<td>3960</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Nunc OmniTray (rectangular plates), case of 60</td>
			<td>Thermo Scientific Rochester</td>
			<td>242811</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Rectangular Ice Pan, Midi (4L)</td>
			<td>Corning Life Sciences</td>
			<td>432104</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Secure-Gard Cone Mask, case of 300</td>
			<td>Cardinal Health</td>
			<td>AT7509</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>AluminaSeal, pack of 100</td>
			<td>Diversified Biotech</td>
			<td>ALUM-100</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Breathe-Easy membrane, pack of 100</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sterile, individually wrapped, 50mL Solution Trough/Reagent Reservoir, case of 100</td>
			<td>Sorenson</td>
			<td>S50100</td>
			<td>via Westnet Incorporated</td>
		</tr>
		<tr>
			<td>Plate roller</td>
			<td>VWR</td>
			<td>60941-118</td>
			<td>via VWR</td>
		</tr>
		<tr>
			<td>Cryo Laser Labels - CRYOLAZRTAG 2.64&#34; x 0.277&#34;, pack of 16 sheets</td>
			<td>GA International</td>
			<td>RCL-11T1-WH</td>
			<td>via Labtag.com (template for printing also available from Labtag.com)</td>
		</tr>
		<tr>
			<td>96-well replicator</td>
			<td>V &#38; P Scientific, Inc.</td>
			<td>Custom 407C, 3.18mm pin diameter, 57mm long</td>
			<td>via V &#38; P Scientific, Inc.</td>
		</tr>
		<tr>
			<td>Multitron Pro, 3mm Shaking incubator</td>
			<td>Infors HT</td>
			<td>l10003P</td>
			<td>via Infors HT</td>
		</tr>
		<tr>
			<td>Picus 12 Channel 50-1200&#956;L Electronic Pipette</td>
			<td>Sartorius</td>
			<td>735491PR</td>
			<td>via Sartorius</td>
		</tr>
		<tr>
			<td>Filter Tips 50-1200&#956;L, pack of 960</td>
			<td>Biohit</td>
			<td>14-559-512</td>
			<td>via Fisher Scientific; use electronic multichannel-compatible tips</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td>User-specific vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for Individual Mutant Storage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-130</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Pipettes (P1000, P200, P20, P2)</td>
			<td>Gilson</td>
			<td>F167370</td>
			<td>via Gilson</td>
		</tr>
		<tr>
			<td>Materials for Quality Control PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-130</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>PCR Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnistrips PCR Tubes with domed lids</td>
			<td>Thermo Scientific</td>
			<td>AB0404</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>ART Barrier low-retention pipette tips (10 uL, 100 uL, 1000 uL)</td>
			<td>Molecular BioProducts, Inc.</td>
			<td>Z676543 (10 uL), Z676713 (100 uL), Z676802 (1000 uL)</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Pipettes (P1000, P200, P20, P2)</td>
			<td>Gilson</td>
			<td>F167370</td>
			<td>via Gilson</td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-130</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>MasterPure DNA Purification Kit</td>
			<td>Epicentre</td>
			<td>MCD85201</td>
			<td>via Epicentre Technologies Corp</td>
		</tr>
		<tr>
			<td>GeneRuler 1 kb Plus DNA Ladder, ready-to-use</td>
			<td>Thermo Scientific</td>
			<td>SM1333</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>RediLoad Loading Buffer</td>
			<td>Invitrogen</td>
			<td>750026</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals for Library and Individual Mutant Storage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol MB Grade, 1L</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td></td>
			<td></td>
			<td>Per 1L dH2O: 10g tryptone, 5g yeast extract, 5g NaCl, 1ml 1N NaOH (Current Protocols in Molecular Biology.&#160; Wiley, 1994.)</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Sigma Aldrich</td>
			<td>T7293</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Sigma Aldrich</td>
			<td>Y1625</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S8045</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>LB&#160; agar</td>
			<td></td>
			<td></td>
			<td>See preparation above, add 15g Bacto Agar</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Sigma Aldrich</td>
			<td>A5306</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Gentamicin sulfate, 10g</td>
			<td>BioReagent</td>
			<td>1405-41-0</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Gibco</td>
			<td>11815024</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Ethanol, 190 proof</td>
			<td>Decon</td>
			<td>04-355-221</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Chemicals for Quality Control PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>User-preferred vendor</td>
			<td></td>
			<td>See primers listed in Table 3</td>
		</tr>
		<tr>
			<td>Corning cellgro Molecular Biology Grade Water</td>
			<td>Corning</td>
			<td>46000CV</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Taq Polymerase Buffer</td>
			<td>Invitrogen</td>
			<td>10342020</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase, recombinant</td>
			<td>Invitrogen</td>
			<td>10342020</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Invitrogen</td>
			<td>10297018</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td>via Sigma-Aldrich</td>
		</tr>
	</tbody>
</table>

replication of the ordered, nonredundant library of pseudomonas aeruginosa strain pa14 transposon insertion mutants

在人类环境中, 铜绿假单胞菌是一种表型、genotypically 多样、适应性强的革兰阴性菌。铜绿假单胞菌能够形成生物膜, 发展耐药性, 产生致病因素, 并在慢性感染过程中迅速发展。因此, 铜绿假单胞菌可以引起急性和慢性, 难以治疗感染, 导致在某些病人的严重发病率。铜绿假单胞菌菌株 PA14 是一个具有保守基因组结构的人的临床隔离, 它感染了各种哺乳动物和 nonvertebrate 宿主, 使 PA14 成为研究这种病原体的一个诱人的菌株。在 2006年, 非冗余座子插入突变体库包含5459变种对应于4596预测 PA14 基因的产生。此后, PA14 图书馆的分布使研究界能够更好地了解个体基因的功能和铜绿假单胞菌的复杂通路。通过复制过程维护库完整性需要正确的处理和精确的技术。为此, 本手稿提出的协议, 详细描述了涉及的步骤, 图书馆复制, 图书馆质量控制和适当储存的个别变种人。

十二 PA14NR 集的新副本是使用协议 I 复制的, 并使用第三号议定书对生成的新副本进行了质量控制评估。
PA14NR 设置突变板连同控制板, 包括野生型 PA14 接种和适合井插在预设模式 (图 4A), 复制后方法在协议 i. 控制板包括在 PA14NR 设置, 以评估可能的井间踪污染时执行板块复制。此外, 控制板可以用来练习复?...

Watch this Scientific Journal Video about Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants at JoVE.com

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

铜绿假单胞菌感染会导致易受害主机发病率显著。非冗余座子插入突变库的绿脓杆菌菌株 PA14, 指定为 PA14NR 集, 促进分析的基因功能在许多过程中。这里提出了一个协议, 以生成高质量的副本 PA14NR 集突变库。

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

replication-ordered-nonredundant-library-pseudomonas-aeruginosa

Research

JoVE Journal

Immunology and Infection

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

8.0K 次观看.  Massachusetts General Hospital. 铜绿假单胞菌感染会导致易受害主机发病率显著。非冗余座子插入突变库的绿脓杆菌菌株 PA14, 指定为 PA14NR 集, 促进分析的基因功能在许多过程中。这里提出了一个协议, 以生成高质量的副本 PA14NR 集突变库。

视频: Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

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 ...

科研

遗传学

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

多拷贝产质粒在抗生素耐药性演变中的作用试验

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

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 ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

在伴有海藻酸钠生产新基因的鉴定<em&gt;铜绿假单胞菌</em&gt;使用小型<em&gt; himar1</em&gt;水手转座子介导的诱变

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

免疫与感染

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

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 outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

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

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

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 ...

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 ...

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 ...

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 ...