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可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

Published: September 01, 2018
doi:

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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23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57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, 一个删除的的基因是证明。多编码的外层膜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的删除对两个三联 autotransporter adhesins (TAAs)、合成耶尔森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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