可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失
Özet
在这里, 我们提出了使用预先存在的抗生素耐药性-盒式删除结构作为一个基础, 使删除突变体在其他大肠杆菌菌株。这种删除突变可以调动和插入到相应的受体菌株的位置使用 P1 噬菌体转导。
Abstract
第一种研究未知基因在细菌中的作用的方法是创建一个基因的敲除。在这里, 我们描述了一个健壮和快速的协议, 将基因缺失突变从一个大肠杆菌的菌株转移到另一个, 利用广义转导与噬菌体 P1。这种方法要求突变是可选择的 (例如,基于基因中断使用抗生素盒插入)。这种抗生素盒可以从捐献者的应变中调动出来, 并引入一个感兴趣的受体菌株, 迅速而容易地产生基因删除突变体。该抗生素盒可以设计为包括 flippase 识别网站, 允许通过特定地点的 recombinase 切除卡带, 以产生一个干净的敲出, 只有一个〜100基对长的疤痕序列在基因组。我们通过敲除 autotransporter 合成中涉及的一个装配因子的多摩基因来证明该协议, 并测试这一击出对两三联 autotransporter adhesins 合成和功能的影响。虽然 P1 转导基因的缺失有其局限性, 但其实施的简便性和速度使其成为其他基因缺失的一种有吸引力的替代方法。
Introduction
研究基因功能的一个共同的第一种方法是执行敲出诱变和观察结果表型。这也被称为反向遗传学。细菌大肠杆菌一直是分子生物学的主力, 在过去的70年左右, 由于它的培养和它的顺从的遗传操作1。在大肠杆菌中产生基因缺失的方法有好几种, 包括标记交换诱变2、3和最近的重组工程使用λ红色或 Rac ET 系统4,5,6。
在广泛使用的系统中, 单个基因的编码序列被一种抗生素耐药性盒取代, 以后可以从5、7号染色体中切除。编码序列被替换, 例如由卡那霉素 (菅直人) 抵抗卡带, 由 flippase (FLP) 识别目标 (首次登记) 站点在两边旁边。首次登记税的地点是由 recombinase FLP 认可的, 该处介导在首次登记税地点之间, 导致删除菅直人卡带的地点特定的重组。这样, 可以实现对给定基因编码序列的完全删除, 只留下大约100基对 (bp) 的最小疤痕序列 (图 1)。
就在十年前, 所谓的京王收藏被开发出来了。这是一个基于标准实验室大肠杆菌K12 菌株的细菌库, 几乎所有非必需基因都被λ红色重组7,8单独删除。此集合中的克隆每个都有一个编码序列, 替换为可抽税的坎电阻盒。在许多应用中, 京王系列已被证明是一个有用的工具9。其中一个应用是在其他大肠杆菌菌株中产生删除突变体。从一个给定的删除克隆的菅直人可以动员一般传感噬菌体, 如 P110,11,12,13,14。从这种菌株中制备的噬菌体可以用来感染受体大肠杆菌, 在低但可靠的频率下, 可以通过同源重组将菅直人盒包含的区域纳入受体基因组。(图 2)。Transductants 可以选择在含菅直人的培养基上生长。在此之后, 如果需要去除抗生素耐药性盒, FLP recombinase 可以提供给 transductant 菌株的反式。对含有氨苄西林 (amp) 抗性标记物的 FLP 质粒进行了筛选, 对其进行了筛查, 并对野型编码序列和菅直人盒的正确切除进行了菌落 PCR 验证。
在这里, 提出了一个详细的协议, 描述了根据上述战略生产出的大肠杆菌菌株的每一个步骤。作为一个例子, 一个删除的摩的基因是证明。多摩编码的外层膜β-桶蛋白是运输和组装模块 (TAM) 的一部分, 涉及某些 autotransporter 蛋白的合成和毛菌15,16,17。然后用这个敲出的菌株来考察多摩的删除对两个三联 autotransporter adhesins (TAAs)、合成耶尔森氏杆菌和大肠杆菌免疫球蛋白 (Ig) 结合黏附 TAA的影响。18,19。
Protocol
1. 菌株和质粒 菌株 使用大肠杆菌菌株 BW251135, JW4179 (BW25113摩:: 菅直人)7, BL21 (DE3)20, BL21ΔABCF21。有关详细信息, 请参阅材料表。 噬菌体 使用噬菌体 P1vir的一般转导。将噬菌体储存在少量氯仿滴下的液体储存中 (见步骤 2.2)。有关…
Representative Results
多摩BL21ΔABCF 的产生: 上面概述的战略以前曾被用来生产 BL21 (DE3) 的衍生物菌株, 这是用于蛋白质生产的标准实验室菌株, 它被优化用于外膜蛋白生产, 并被称为 BL21ΔABCF21。这种菌株缺乏四基因编码的丰富的外膜蛋白, 因此, 能够产生更多的 heterologously 表达外膜蛋白比野生型菌株。为了测试 TAM 是否参与了 TA…
Discussion
P1 转导是一种快速、稳健、可靠的方法, 可在大肠杆菌中产生基因缺失。这在这里展示了传感一个摩中的删除突变从一个王庆会捐献者的应变 BL21-derived 接受。转导过程的主要阶段是传感裂解液的产生、转导本身、菅直人抗性盒的切除以及 PCR 的验证。总的来说, 这个过程需要大约1周的时间, 不需要使用分子生物学方法, 除了最终的 PCR 进行验证。因此, P1 转导可以在花费的努力和时间?…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
京王收集菌株来自国家 BioResource 项目 ( 日本 ) :大肠杆菌。我们感谢德克左翼 (奥斯陆大学生物科学系) 继续提供支持。这项工作由挪威青年研究员补助金 249793 (对杰克. 里奥) 的研究理事会资助。
Materials
| Strains | |||
| E. coli BW25113 | NIG | ME6092 | Wild-type strain of Keio collection |
| E. coli BL21(DE3) | Merck | 69450-3 | Expression strain |
| E. coli BL21DABCF | Addgene | 102270 | Derived from BL21(DE3) |
| E. coli JW4179 | NIG | JW4179-KC | tamA deletion mutant |
| P1 vir | NIG | HR16 | Generally transducing bacteriophage |
| Plasmids | |||
| pCP20 | CGSC | 14177 | conditionally replicating plasmid with FLP |
| pASK-IBA2 | IBA GmbH | 2-1301-000 | expression vector |
| pEibD10 | N/A | N/A | for production of EibD; plasmid available on request |
| pET22b+ | Merck | 69744-3 | expression vector |
| pIBA2-YadA | N/A | N/A | for production of YadA; plasmid available on request |
| Chemicals | |||
| Acetic acid | ThermoFisher | 33209 | |
| Agar | BD Bacto | 214010 | |
| Agarose | Lonza | 50004 | |
| Ampicillin | Applichem | A0839 | |
| Anhydrotetracycline | Abcam | ab145350 | |
| anti-collagen type I antibody COL-1 | Sigma | C2456 | |
| Bovine collagen type I | Sigma | C9791 | |
| Calcium chloride | Merck | 102382 | |
| Chloroform | Merck | 102445 | |
| Di-sodium hydrogen phosphate | VWR | 28029 | |
| DNA dye | Thermo | S33102 | |
| DNA molecular size marker | New England BioLabs | N3232S | |
| DNase I | Sigma | DN25 | |
| dNTP mix | New England Biolabs | N0447 | |
| ECL HRP substrate | Advansta | K-12045 | |
| EDTA | Applichem | A2937 | |
| Glycerol | VWR | 24388 | |
| goat anti-mouse IgG-HRP | Santa Cruz | sc-2005 | |
| goat anti-rabbit IgG-HRP | Agrisera | AS10668 | |
| HEPES | VWR | 30487 | |
| Isopropyl thiogalactoside | VWR | 43714 | |
| Kanamycin | Applichem | A1493 | |
| Lysozyme | Applichem | A4972 | |
| Magnesium chloride | VWR | 25108 | |
| Manganese chloride | Sigma | 221279 | |
| N-lauroyl sarcosine | Sigma | L9150 | |
| Skim milk powder | Sigma | 70166 | |
| Sodium chloride | VWR | 27808 | |
| tamA forward primer | Invitrogen | N/A | Sequence 5'-GAAAAAAGGATATTCAGGAGAAAATGTG-3' |
| tamA reverse primer | Invitrogen | N/A | Sequence 5'-TCATAATTCTGGCCCCAGACC-3' |
| Taq DNA polymerase | New England Biolabs | M0267 | |
| Tri-sodium citrate | Merck | 106448 | |
| Tryptone | VWR | 84610 | |
| Tween20 | Sigma | P1379 | |
| Yeast extract | Merck | 103753 | |
| Equipment | |||
| Agarose gel electrophoresis chamber | Hoefer | SUB13 | |
| Bead beater | Thermo | FP120A-115 | |
| CCD camera | Kodak | 4000R | |
| Electroporation cuvettes | Bio-Rad | 165-2089 | |
| Electroporation unit | Bio-Rad | 1652100 | |
| Gel imager | Nippon Genetics | GP-03LED | |
| Incubating shaker | Infors HT | Minitron | |
| Incubator | VWR | 390-0482 | |
| Microcentrifuge | Eppendorf | 5415D | |
| Microwave oven | Samsung | CM1099A | |
| PCR machine | Biometra | Tpersonal | |
| PCR strips | Axygen | PCR-0208-CP-C | |
| pH meter | Hanna Instruments | HI2211-01 | |
| PVDF membrane | ThermoFisher | 88518 | |
| SDS-PAG electrophoresis chamber | ThermoFisher | A25977 | |
| Tabletop centrifuge | Beckman Coulter | B06322 | |
| Vortex mixer | Scientific Industries | SI-0236 | |
| Water bath | GFL | D3006 | |
| Wet transfer unit | Hoefer | TE22 |
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Bu Makaleden Alıntı Yapın
Saragliadis, A., Trunk, T., Leo, J. C. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. J. Vis. Exp. (139), e58267, doi:10.3791/58267 (2018).