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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Materials for Library Replication | |||
| Sterile 96-well Tissue-culture treated, case of 50 | Corning Life Sciences | 353072 | via Fisher Scientific |
| Sterile 96 Well Clear V-Bottom 2000μL Deep Well Plates, case of 25 | Corning Life Sciences | 3960 | via Fisher Scientific |
| Nunc OmniTray (rectangular plates), case of 60 | Thermo Scientific Rochester | 242811 | via Fisher Scientific |
| Rectangular Ice Pan, Midi (4L) | Corning Life Sciences | 432104 | via Fisher Scientific |
| Secure-Gard Cone Mask, case of 300 | Cardinal Health | AT7509 | via Fisher Scientific |
| AluminaSeal, pack of 100 | Diversified Biotech | ALUM-100 | via Fisher Scientific |
| Breathe-Easy membrane, pack of 100 | Diversified Biotech | BEM-1 | via Sigma-Aldrich |
| Sterile, individually wrapped, 50mL Solution Trough/Reagent Reservoir, case of 100 | Sorenson | S50100 | via Westnet Incorporated |
| Plate roller | VWR | 60941-118 | via VWR |
| Cryo Laser Labels – CRYOLAZRTAG 2.64" x 0.277", pack of 16 sheets | GA International | RCL-11T1-WH | via Labtag.com (template for printing also available from Labtag.com) |
| 96-well replicator | V & P Scientific, Inc. | Custom 407C, 3.18mm pin diameter, 57mm long | via V & P Scientific, Inc. |
| Multitron Pro, 3mm Shaking incubator | Infors HT | l10003P | via Infors HT |
| Picus 12 Channel 50-1200μL Electronic Pipette | Sartorius | 735491PR | via Sartorius |
| Filter Tips 50-1200μL, pack of 960 | Biohit | 14-559-512 | via Fisher Scientific; use electronic multichannel-compatible tips |
| Dry Ice | User-specific vendor | ||
| Materials for Individual Mutant Storage | |||
| Fisherbrand Premium Microcentrifuge Tubes: 1.5mL | Fisher Scientific | 05-408-130 | via Fisher Scientific |
| Pipettes (P1000, P200, P20, P2) | Gilson | F167370 | via Gilson |
| Materials for Quality Control PCR | |||
| Fisherbrand Premium Microcentrifuge Tubes: 1.5mL | Fisher Scientific | 05-408-130 | via Fisher Scientific |
| NanoDrop | Thermo Scientific | ND-2000 | via ThermoFisher |
| PCR Thermocycler | |||
| Omnistrips PCR Tubes with domed lids | Thermo Scientific | AB0404 | via Fisher Scientific |
| ART Barrier low-retention pipette tips (10 uL, 100 uL, 1000 uL) | Molecular BioProducts, Inc. | Z676543 (10 uL), Z676713 (100 uL), Z676802 (1000 uL) | via Sigma-Aldrich |
| Pipettes (P1000, P200, P20, P2) | Gilson | F167370 | via Gilson |
| Fisherbrand Premium Microcentrifuge Tubes: 1.5mL | Fisher Scientific | 05-408-130 | via Fisher Scientific |
| MasterPure DNA Purification Kit | Epicentre | MCD85201 | via Epicentre Technologies Corp |
| GeneRuler 1 kb Plus DNA Ladder, ready-to-use | Thermo Scientific | SM1333 | via ThermoFisher |
| RediLoad Loading Buffer | Invitrogen | 750026 | via ThermoFisher |
| Chemicals | |||
| Chemicals for Library and Individual Mutant Storage | |||
| Glycerol MB Grade, 1L | Sigma Aldrich | G5516 | via Sigma-Aldrich |
| LB Broth | Per 1L dH2O: 10g tryptone, 5g yeast extract, 5g NaCl, 1ml 1N NaOH (Current Protocols in Molecular Biology. Wiley, 1994.) | ||
| Tryptone | Sigma Aldrich | T7293 | via Sigma-Aldrich |
| Yeast Extract | Sigma Aldrich | Y1625 | via Sigma-Aldrich |
| Sodium Chloride | Sigma Aldrich | S7653 | via Sigma-Aldrich |
| Sodium Hydroxide | Sigma Aldrich | S8045 | via Sigma-Aldrich |
| LB agar | See preparation above, add 15g Bacto Agar | ||
| Bacto Agar | Sigma Aldrich | A5306 | via Sigma-Aldrich |
| Gentamicin sulfate, 10g | BioReagent | 1405-41-0 | via Sigma-Aldrich |
| Kanamycin sulfate | Gibco | 11815024 | via ThermoFisher |
| Ethanol, 190 proof | Decon | 04-355-221 | via Fisher Scientific |
| Chemicals for Quality Control PCR | |||
| Primers | User-preferred vendor | See primers listed in Table 3 | |
| Corning cellgro Molecular Biology Grade Water | Corning | 46000CV | via Fisher Scientific |
| Taq Polymerase Buffer | Invitrogen | 10342020 | via ThermoFisher |
| Taq DNA Polymerase, recombinant | Invitrogen | 10342020 | via ThermoFisher |
| dNTPs | Invitrogen | 10297018 | via ThermoFisher |
| Agarose | Sigma | A9539 | via Sigma-Aldrich |
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