BioVector? pJOE8999枯草芽孢桿菌CRISPR-Cas9基因編輯質(zhì)粒pJOE8999 Plasmid 數(shù)據(jù)說明書

產(chǎn)品名稱: BioVector? pJOE8999 質(zhì)粒
中文名稱: BioVector? pJOE8999 枯草芽孢桿菌CRISPR-Cas9基因編輯質(zhì)粒
英文名稱: BioVector? pJOE8999 Bacillus subtilis CRISPR-Cas9 Genome Editing Plasmid
產(chǎn)品類別: CRISPR-Cas9基因編輯載體；穿梭載體
質(zhì)粒類型: 大腸桿菌-枯草芽孢桿菌穿梭載體；溫度敏感型基因編輯載體
用途: 用于枯草芽孢桿菌及其他革蘭氏陽性菌的基因組編輯，包括基因敲除、點(diǎn)突變引入、大片段缺失等
來源信息: 由德國斯圖加特大學(xué) Josef Altenbuchner 教授于2016年構(gòu)建并發(fā)表
載體大小: 7794 bp
復(fù)制子:

• 大腸桿菌復(fù)制子：pUC 最小復(fù)制起點(diǎn)

• 枯草芽孢桿菌復(fù)制子：rep pE194ts (溫度敏感型復(fù)制起點(diǎn))
  篩選標(biāo)記: 卡那霉素抗性基因 (Kanamycin resistance)，在大腸桿菌和枯草芽孢桿菌中均可使用
  抗性濃度:

• 大腸桿菌：30-50 μg/mL 卡那霉素

• 枯草芽孢桿菌：5 μg/mL 卡那霉素
  啟動(dòng)子:

• PmanP: 枯草芽孢桿菌甘露糖誘導(dǎo)型啟動(dòng)子，用于驅(qū)動(dòng) cas9 基因表達(dá)。甘露糖存在時(shí)表達(dá)量可提高 4-7 倍。該啟動(dòng)子在大腸桿菌中無活性

• PvanP*: 半合成啟動(dòng)子，用于驅(qū)動(dòng) sgRNA 轉(zhuǎn)錄，位于 lacZ α 片段下游
  sgRNA 克隆位點(diǎn): 兩個(gè) BsaI 限制性內(nèi)切酶位點(diǎn)，用于插入 20 nt 的靶向間隔序列；可替換 lacZ α 片段實(shí)現(xiàn)藍(lán)白斑篩選
  同源臂克隆位點(diǎn): 兩個(gè)相鄰的 SfiI 位點(diǎn)，用于順序整合左右同源臂
  特性特征:

1. 溫度敏感性復(fù)制: 枯草芽孢桿菌中，在 30°C 允許溫度下質(zhì)?？蓮?fù)制；在 37°C 及以上非允許溫度下復(fù)制被抑制，用于篩選同源重組整合事件

2. 甘露糖誘導(dǎo)表達(dá): cas9 基因在 PmanP 啟動(dòng)子控制下，添加 0.2% 甘露糖可誘導(dǎo) Cas9 蛋白表達(dá)，增強(qiáng)編輯效率

3. 單質(zhì)粒系統(tǒng): Cas9 和 sgRNA 均位于同一質(zhì)粒上，操作簡便

4. 高效編輯: 無需篩選標(biāo)記即可高效獲得編輯突變體，適用于多種枯草芽孢桿菌菌株

5. 宿主范圍: 可擴(kuò)展應(yīng)用于其他革蘭氏陽性菌，如蘇云金芽孢桿菌、炭疽芽孢桿菌等
   操作流程:

6. 通過 BsaI 位點(diǎn)克隆 20 nt 靶向間隔序列

7. 通過 SfiI 位點(diǎn)克隆兩側(cè)同源臂

8. 轉(zhuǎn)化枯草芽孢桿菌感受態(tài)細(xì)胞

9. 甘露糖誘導(dǎo) Cas9 表達(dá)，產(chǎn)生 DNA 雙鏈斷裂

10. 同源重組修復(fù)，實(shí)現(xiàn)基因組編輯

11. 高溫傳代消除質(zhì)粒
    質(zhì)控載體: BioVector? pUC19 質(zhì)粒
    宿主菌: BioVector? DH5α 化學(xué)感受態(tài)細(xì)胞（用于質(zhì)粒擴(kuò)增）
    質(zhì)量控制: 測(cè)序驗(yàn)證：100%正確；無菌檢測(cè)：陰性；酶切鑒定：符合預(yù)期條帶
    儲(chǔ)存與運(yùn)輸: -20°C 或干冰運(yùn)輸與保存
    僅供科研使用

English:
BioVector? pJOE8999 Plasmid Datasheet

Product Name: BioVector? pJOE8999 Plasmid
Chinese Name: BioVector? pJOE8999 枯草芽孢桿菌CRISPR-Cas9基因編輯質(zhì)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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