pKOR1 BioVector®金黄色葡萄球菌等位基因置换质粒载体Staphylococcus aureus Allelic Replacement Plasmid

BioVector® pKOR1 金黄色葡萄球菌等位基因置换质粒 / Staphylococcus aureus Allelic Replacement Plasmid 数据说明书

产品名称: BioVector® pKOR1 质粒
同义词: BioVector® pKOR1 穿梭载体
中文名称: BioVector® pKOR1 金黄色葡萄球菌等位基因置换质粒
英文名称: BioVector® pKOR1 Staphylococcus aureus Allelic Replacement Plasmid
产品类别: 穿梭载体；基因敲除质粒
质粒类型: 大肠杆菌-金黄色葡萄球菌穿梭载体；温度敏感型等位基因置换载体
用途: 用于金黄色葡萄球菌染色体基因的无标记等位基因置换突变；快速克隆与反筛选选择
来源信息: 由Taeok Bae 和 Olaf Schneewind 构建并馈赠
载体大小: 9974 bp
复制子:

• 大肠杆菌复制子：ColE1/pMB1/pBR322 高拷贝数复制起点

• 金黄色葡萄球菌复制子：pAMα1 (温度敏感型)
  抗性筛选标记:

• 大肠杆菌筛选：氨苄青霉素 (100 μg/mL)

• 金黄色葡萄球菌筛选：氯霉素 (25 μg/mL)
  克隆方法: Gateway 克隆 (λ 重组)；携带 ccdB 致死基因用于阳性克隆筛选
  基因元件:

1. ccdB 基因: 编码 DNA 促旋酶毒性蛋白，用于正向选择；仅在宿主菌 DB3.1 中可稳定维持

2. attP1/attP2 位点: Gateway 克隆系统重组位点

3. Pxyl/tetO 启动子: 受脱水四环素诱导的反向选择启动子

4. secY 反义转录本: 脱水四环素诱导后表达，抑制金黄色葡萄球菌生长，用于反向选择

5. TetR: 四环素阻遏蛋白编码基因
   

6. 

7. 特性特征:

8. 温度敏感性复制: 在 30-37°C 条件下可在金黄色葡萄球菌中复制；在 43°C 非允许温度下复制被抑制，用于筛选同源重组整合事件

9. 两步筛选机制:

   • 第一步 (整合)：43°C 培养选择质粒通过同源重组整合入染色体

   • 第二步 (切除)：脱水四环素诱导 secY 反义 RNA 表达，抑制细胞生长，选择质粒切除和丢失事件

10. 高效性: 无需抗生素标记即可高频率获得等位基因置换突变体

11. 应用: 广泛应用于金黄色葡萄球菌的基因敲除、等位基因置换、点突变引入研究
    细菌培养条件:

• 大肠杆菌宿主菌：BioVector® DB3.1 感受态细胞 (用于 pKOR1 扩增)

• 金黄色葡萄球菌宿主菌：BioVector® RN4220 或相应临床分离株

• 培养温度：30-37°C (质粒复制)；43°C (整合筛选)
  诱导条件: 脱水四环素 (终浓度根据实验优化)
  质控载体: BioVector® pUC19 质粒
  宿主菌: BioVector® DB3.1 化学感受态细胞 (用于质粒扩增)
  质量控制: 测序验证：100%正确；无菌检测：阴性；酶切鉴定：符合预期条带
  储存与运输: -20°C 或干冰运输与保存
  仅供科研使用

English:
BioVector® pKOR1 Staphylococcus aureus Allelic Replacement Plasmid Datasheet

Product Name: BioVector® pKOR1 Plasmid
Synonyms: BioVector® pKOR1 Shuttle Vector
Chinese Name: BioVector® pKOR1 金黄色葡萄球菌等位基因置换质粒
English Name: BioVector® pKOR1 Staphylococcus aureus Allelic Replacement Plasmid
Product Category: Shuttle Vector; Gene Knockout Plasmid
Plasmid Type: E. coli-S. aureus Shuttle Vector; Temperature-Sensitive Allelic Replacement Vector
Purpose: Unmarked allelic replacement mutagenesis of S. aureus chromosomal genes; rapid cloning via lambda recombination and counter-selection
Source: Constructed and gifted by Taeok Bae and Olaf Schneewind
Vector Size: 9974 bp
Replicons:

• E. coli Replicon: ColE1/pMB1/pBR322 high-copy origin of replication

• S. aureus Replicon: pAMα1 (temperature-sensitive)
  Selection Markers:

• E. coli Selection: Ampicillin (100 μg/mL)

• S. aureus Selection: Chloramphenicol (25 μg/mL)
  Cloning Method: Gateway Cloning (λ recombination); ccdB lethal gene for positive selection
  Genetic Elements:

1. ccdB Gene: Encodes DNA gyrase toxin protein for positive selection; only maintained in DB3.1 host strain

2. attP1/attP2 Sites: Gateway cloning system recombination sites

3. Pxyl/tetO Promoter: Counter-selection promoter inducible by anhydrotetracycline

4. secY Antisense Transcript: Expressed upon anhydrotetracycline induction, inhibits S. aureus growth for counter-selection

5. TetR: Tetracycline repressor protein coding gene
   Characteristics:

6. Temperature-Sensitive Replication: Replicates in S. aureus at 30-37°C; replication is inhibited at the non-permissive temperature of 43°C, allowing selection for homologous recombination integration events

7. Two-Step Selection Mechanism:

   • Step 1 (Integration): Selection at 43°C for plasmid integration into the chromosome via homologous recombination

   • Step 2 (Excision): Anhydrotetracycline induction of secY antisense RNA expression inhibits cell growth, selecting for plasmid excision and loss events

8. High Efficiency: Allelic replacement mutants can be generated at high frequency without the need for antibiotic marker selection

9. Applications: Widely used for gene knockout, allelic replacement, and point mutation introduction in Staphylococcus aureus
   Bacterial Growth Conditions:

• E. coli Host Strain: BioVector® DB3.1 Competent Cells (for pKOR1 amplification)

• S. aureus Host Strain: BioVector® RN4220 or relevant clinical isolates

• Growth Temperature: 30-37°C (plasmid replication); 43°C (integration selection)
  Induction Condition: Anhydrotetracycline (final concentration optimized experimentally)
  Control Vector: BioVector® pUC19 Plasmid
  Host Strain: BioVector® DB3.1 Chemically Competent Cells (for plasmid amplification)
  Quality Control: Sequencing Validation: 100% correct; Sterility: Negative; Restriction Digestion: Expected bands
  Storage and Shipment: Stored and shipped at -20°C or on dry ice
  For Research Use Only

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