pJOE8999 BioVector®枯草芽孢杆菌CRISPR-Cas9基因编辑质粒pJOE8999 Plasmid BioVector NTCC典型培养物保藏中心

BioVector® pJOE8999枯草芽孢杆菌CRISPR-Cas9基因编辑质粒pJOE8999 Plasmid 数据说明书

产品名称: BioVector® pJOE8999 质粒
中文名称: BioVector® pJOE8999 枯草芽孢杆菌CRISPR-Cas9基因编辑质粒
英文名称: BioVector® pJOE8999 Bacillus subtilis CRISPR-Cas9 Genome Editing Plasmid
产品类别: CRISPR-Cas9基因编辑载体；穿梭载体
质粒类型: 大肠杆菌-枯草芽孢杆菌穿梭载体；温度敏感型基因编辑载体
用途: 用于枯草芽孢杆菌及其他革兰氏阳性菌的基因组编辑，包括基因敲除、点突变引入、大片段缺失等
来源信息: 由德国斯图加特大学 Josef Altenbuchner 教授于2016年构建并发表
载体大小: 7794 bp
复制子:

• 大肠杆菌复制子：pUC 最小复制起点

• 枯草芽孢杆菌复制子：rep pE194ts (温度敏感型复制起点)
  筛选标记: 卡那霉素抗性基因 (Kanamycin resistance)，在大肠杆菌和枯草芽孢杆菌中均可使用
  抗性浓度:

• 大肠杆菌：30-50 μg/mL 卡那霉素

• 枯草芽孢杆菌：5 μg/mL 卡那霉素
  启动子:

• PmanP: 枯草芽孢杆菌甘露糖诱导型启动子，用于驱动 cas9 基因表达。甘露糖存在时表达量可提高 4-7 倍。该启动子在大肠杆菌中无活性

• PvanP*: 半合成启动子，用于驱动 sgRNA 转录，位于 lacZ α 片段下游
  sgRNA 克隆位点: 两个 BsaI 限制性内切酶位点，用于插入 20 nt 的靶向间隔序列；可替换 lacZ α 片段实现蓝白斑筛选
  同源臂克隆位点: 两个相邻的 SfiI 位点，用于顺序整合左右同源臂
  特性特征:

1. 温度敏感性复制: 枯草芽孢杆菌中，在 30°C 允许温度下质粒可复制；在 37°C 及以上非允许温度下复制被抑制，用于筛选同源重组整合事件

2. 甘露糖诱导表达: cas9 基因在 PmanP 启动子控制下，添加 0.2% 甘露糖可诱导 Cas9 蛋白表达，增强编辑效率

3. 单质粒系统: Cas9 和 sgRNA 均位于同一质粒上，操作简便

4. 高效编辑: 无需筛选标记即可高效获得编辑突变体，适用于多种枯草芽孢杆菌菌株

5. 宿主范围: 可扩展应用于其他革兰氏阳性菌，如苏云金芽孢杆菌、炭疽芽孢杆菌等
   操作流程:

6. 通过 BsaI 位点克隆 20 nt 靶向间隔序列

7. 通过 SfiI 位点克隆两侧同源臂

8. 转化枯草芽孢杆菌感受态细胞

9. 甘露糖诱导 Cas9 表达，产生 DNA 双链断裂

10. 同源重组修复，实现基因组编辑

11. 高温传代消除质粒
    质控载体: BioVector® pUC19 质粒
    宿主菌: BioVector® DH5α 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® pJOE8999 Plasmid Datasheet

Product Name: BioVector® pJOE8999 Plasmid
Chinese Name: BioVector® pJOE8999 枯草芽孢杆菌CRISPR-Cas9基因编辑质粒
English Name: BioVector® pJOE8999 Bacillus subtilis CRISPR-Cas9 Genome Editing Plasmid
Product Category: CRISPR-Cas9 Genome Editing Vector; Shuttle Vector
Plasmid Type: E. coli-B. subtilis Shuttle Vector; Temperature-Sensitive Genome Editing Vector
Purpose: Genome editing in Bacillus subtilis and other Gram-positive bacteria, including gene knockout, point mutation introduction, and large fragment deletion
Source: Constructed and published by Professor Josef Altenbuchner, University of Stuttgart, Germany, in 2016
Vector Size: 7794 bp
Replicons:

• E. coli Replicon: pUC minimal origin of replication

• B. subtilis Replicon: rep pE194ts (temperature-sensitive origin of replication)
  Selection Marker: Kanamycin resistance gene, functional in both E. coli and B. subtilis
  Resistance Concentrations:

• E. coli: 30-50 μg/mL kanamycin

• B. subtilis: 5 μg/mL kanamycin
  Promoters:

• PmanP: B. subtilis mannose-inducible promoter driving cas9 expression. Expression increases 4-7 fold in the presence of mannose. This promoter is not active in E. coli

• PvanP*: Semi-synthetic promoter driving sgRNA transcription, located downstream of the lacZ α fragment
  sgRNA Cloning Site: Two BsaI restriction sites for inserting the 20 nt targeting spacer sequence; replacement of lacZ α fragment enables blue-white screening
  Homology Arm Cloning Site: Two adjacent SfiI sites for ordered integration of left and right homology arms
  Characteristics:

1. Temperature-Sensitive Replication: In B. subtilis, the plasmid replicates at the permissive temperature (30°C); replication is inhibited at non-permissive temperatures (≥37°C), allowing selection for homologous recombination integration events

2. Mannose-Inducible Expression: The cas9 gene is under the control of the PmanP promoter; addition of 0.2% mannose induces Cas9 protein expression, enhancing editing efficiency

3. Single-Plasmid System: Both Cas9 and sgRNA are located on the same plasmid, simplifying operation

4. High Efficiency: Editable mutants can be obtained at high frequency without the need for selection markers; suitable for various B. subtilis strains

5. Host Range: Extendable to other Gram-positive bacteria such as Bacillus thuringiensis and Bacillus anthracis
   Procedure:

6. Clone 20 nt targeting spacer sequence via BsaI sites

7. Clone homology arms via SfiI sites

8. Transform into B. subtilis competent cells

9. Induce Cas9 expression with mannose to generate DNA double-strand breaks

10. Homologous recombination repair enables genome editing

11. Cure plasmid through high-temperature passaging
    Control Vector: BioVector® pUC19 Plasmid
    Host Strain: BioVector® DH5α 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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