pRK2013Tn5G BioVector®辅助质粒转座子递送质粒载体-BioVector NTCC典型培养物保藏中心

BioVector® pRK2013Tn5G Plasmid

English Version

1. Plasmid Overview

• Plasmid Name: BioVector® pRK2013Tn5G

• Type: Helper plasmid / Transposon delivery plasmid

• Parental Backbone: pRK2013

• Key Application: Used for transposon mutagenesis; mobilizes non-self-transmissible plasmids

• Reference: Nunn and Lory, 1992

2. Genetic Features

• Replicon: ColE1 origin of replication

• Size: Approximately 48 kb (based on pRK2013 backbone)

• Tn5G Transposon: Contains Tn5G, a derivative of the Tn5 transposon used for insertional mutagenesis

• Mobilization Functions: Carries RK2 transfer genes (tra genes), enabling it to function as a helper plasmid for mobilization of non-self-transmissible plasmids in triparental mating experiments

3. Selectable Markers

• Kanamycin Resistance (Kmr): Confers resistance to kanamycin for selection in E. coli and other Gram-negative bacteria

• Gentamicin Resistance (Gmr): Confers resistance to gentamicin, derived from the Tn5G element

• Note: Standard pRK2013 carries only Kmr (Tn903); pRK2013Tn5G is a modified version incorporating Tn5G with Gmr

4. Description and Applications
BioVector® pRK2013Tn5G is a specialized plasmid derived from the well-characterized helper plasmid pRK2013. It contains the Tn5G transposon, which carries a gentamicin resistance marker, allowing for transposon mutagenesis with dual selection capabilities.

The plasmid serves two primary functions:

• Helper Plasmid: Provides RK2 transfer functions in trans to mobilize non-self-transmissible plasmids (e.g., pUCP18 derivatives) from E. coli to recipient Gram-negative bacteria such as Pseudomonas aeruginosa

• Transposon Delivery Vehicle: Delivers Tn5G for random insertional mutagenesis, creating stable mutant libraries with gentamicin resistance as the selectable marker

This plasmid has been widely used in studies involving Pseudomonas aeruginosa and other Gram-negative bacteria for generating mutant strains to study gene function, phage resistance mechanisms, and virulence factors.

5. Mechanism of Action
In triparental mating systems, BioVector® pRK2013Tn5G is transformed into a donor E. coli strain (e.g., HB101). When mixed with a recipient strain (e.g., P. aeruginosa PAK) and another donor strain carrying a non-mobilizable plasmid, pRK2013Tn5G provides the conjugation machinery (Tra functions) to transfer the non-mobilizable plasmid into the recipient. Alternatively, when used alone for transposon mutagenesis, the Tn5G element randomly inserts into the recipient genome, creating gentamicin-resistant mutants.

6. Host Range and Compatibility

• Host Organism: Propagated in Escherichia coli (e.g., K12 HB101)

• Recipient Range: Broad host range; can mobilize plasmids into various Gram-negative bacteria including Pseudomonas, Serratia, Vibrio, and other genera

• Replication: Not maintained in recipients without selection; primarily used as a suicide delivery system for transposon mutagenesis

7. Growth and Culturing Conditions for Host Strain

• Host Strain Carrying Plasmid: BioVector® E. coli K12 HB101 containing pRK2013Tn5G

• Recommended Medium: BioVector® LB-Miller Broth or Agar (high salt)

• Antibiotic Selection: Add kanamycin (50 μg/mL) and/or gentamicin (10-15 μg/mL) as appropriate

• Incubation Conditions: 37°C, aerobic

• Storage: For long-term storage, maintain at -80°C in appropriate medium with 15-20% glycerol

8. Quality Control

• Identity: Verified by restriction digestion pattern and/or sequencing

• Sterility: Negative for contaminants

• Functionality: Confirmed by ability to mobilize test plasmids or generate transposon mutants

9. Applications

• Transposon Mutagenesis: Generation of random insertion mutant libraries in Gram-negative bacteria

• Gene Function Studies: Identification of genes involved in specific phenotypes (phage resistance, biofilm formation, virulence)

• Triparental Mating: Helper plasmid for mobilizing non-self-transmissible plasmids

• Mutant Complementation: Used in conjunction with complementing plasmids to confirm gene function

• Genetic Screens: Identification of genes required for specific environmental adaptations

BioVector® pRK2013Tn5G 质粒说明书

中文版本

1. 质粒概述

• 质粒名称： BioVector® pRK2013Tn5G

• 类型： 辅助质粒 / 转座子递送质粒

• 亲本骨架： pRK2013

• 主要应用： 用于转座子诱变；可 mobilization 非自我传播性质粒

• 参考文献： Nunn and Lory, 1992

2. 遗传特征

• 复制子： ColE1 复制起始位点

• 大小： 约 48 kb（基于 pRK2013 骨架）

• Tn5G 转座子： 含有 Tn5G，Tn5 转座子的衍生版本，用于插入诱变

• mobilization 功能： 携带 RK2 转移基因（tra 基因），使其能够在三亲本杂交实验中作为辅助质粒，用于 mobilization 非自我传播性质粒

3. 选择标记

• 卡那霉素抗性 (Kmr)： 赋予卡那霉素抗性，用于在大肠杆菌和其他革兰氏阴性菌中进行筛选

• 庆大霉素抗性 (Gmr)： 赋予庆大霉素抗性，来源于 Tn5G 元件

• 注意：标准 pRK2013 仅携带 Kmr（Tn903）；pRK2013Tn5G 是整合了带有 Gmr 的 Tn5G 的修饰版本

4. 描述与应用
BioVector® pRK2013Tn5G 是一种源自特征明确的辅助质粒 pRK2013 的专用质粒。它含有携带庆大霉素抗性标记的 Tn5G 转座子，允许进行具有双重筛选能力的转座子诱变。

该质粒具有两个主要功能：

• 辅助质粒： 反式提供 RK2 转移功能，将非自我传播性质粒（例如 pUCP18 衍生物）从大肠杆菌 mobilization 至受体革兰氏阴性菌（如铜绿假单胞菌）

• 转座子递送载体： 递送 Tn5G 进行随机插入诱变，以庆大霉素抗性作为筛选标记，创建稳定的突变体文库

该质粒已广泛用于涉及铜绿假单胞菌和其他革兰氏阴性菌的研究，用于生成突变菌株以研究基因功能、噬菌体抗性机制和毒力因子。

5. 作用机制
在三亲本杂交系统中，BioVector® pRK2013Tn5G 被转化至供体大肠杆菌菌株（如 HB101）。当与受体菌株（如铜绿假单胞菌 PAK）和另一个携带非可 mobilization 性质粒的供体菌株混合时，pRK2013Tn5G 提供接合机制（Tra 功能），将非可 mobilization 性质粒转移至受体中。或者，当单独用于转座子诱变时，Tn5G 元件随机插入受体基因组，产生庆大霉素抗性突变体。

6. 宿主范围与兼容性

• 宿主生物： 在大肠杆菌中扩增（例如 K12 HB101）

• 受体范围： 广泛的宿主范围；可将质粒 mobilization 至多种革兰氏阴性菌，包括假单胞菌属、沙雷氏菌属、弧菌属和其他属

• 复制： 在没有筛选压力的情况下，不在受体中维持；主要用作转座子诱变的自杀性递送系统

7. 宿主菌株的生长与培养条件

• 携带质粒的宿主菌株： 含有 pRK2013Tn5G 的 BioVector® 大肠杆菌 K12 HB101

• 推荐培养基： BioVector® LB-Miller 肉汤或琼脂（高盐）

• 抗生素筛选： 根据需要添加卡那霉素（50 μg/mL）和/或庆大霉素（10-15 μg/mL）

• 培养条件： 37°C，需氧

• 储存： 长期储存时，在含有 15-20% 甘油的适当培养基中保存于 -80°C

8. 质量控制

• 身份鉴定： 通过限制性酶切图谱和/或测序验证

• 无菌检测： 无污染物

• 功能性： 通过 mobilization 测试质粒或产生转座子突变体的能力确认

9. 应用

• 转座子诱变： 在革兰氏阴性菌中生成随机插入突变体文库

• 基因功能研究： 鉴定参与特定表型（噬菌体抗性、生物膜形成、毒力）的基因

• 三亲本杂交： 用于 mobilization 非自我传播性质粒的辅助质粒

• 突变体互补： 与互补质粒联合使用以确认基因功能

• 遗传筛选： 鉴定特定环境适应所需的基因

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