BioVector? pKOR1 金黃色葡萄球菌等位基因置換質(zhì)粒 / Staphylococcus aureus Allelic Replacement Plasmid 數(shù)據(jù)說明書

產(chǎn)品名稱: BioVector? pKOR1 質(zhì)粒
同義詞: BioVector? pKOR1 穿梭載體
中文名稱: BioVector? pKOR1 金黃色葡萄球菌等位基因置換質(zhì)粒
英文名稱: BioVector? pKOR1 Staphylococcus aureus Allelic Replacement Plasmid
產(chǎn)品類別: 穿梭載體；基因敲除質(zhì)粒
質(zhì)粒類型: 大腸桿菌-金黃色葡萄球菌穿梭載體；溫度敏感型等位基因置換載體
用途: 用于金黃色葡萄球菌染色體基因的無標(biāo)記等位基因置換突變；快速克隆與反篩選選擇
來源信息: 由Taeok Bae 和 Olaf Schneewind 構(gòu)建并饋贈
載體大小: 9974 bp
復(fù)制子:

• 大腸桿菌復(fù)制子：ColE1/pMB1/pBR322 高拷貝數(shù)復(fù)制起點(diǎn)

• 金黃色葡萄球菌復(fù)制子：pAMα1 (溫度敏感型)
  抗性篩選標(biāo)記:

• 大腸桿菌篩選：氨芐青霉素 (100 μg/mL)

• 金黃色葡萄球菌篩選：氯霉素 (25 μg/mL)
  克隆方法: Gateway 克隆 (λ 重組)；攜帶 ccdB 致死基因用于陽性克隆篩選
  基因元件:

1. ccdB 基因: 編碼 DNA 促旋酶毒性蛋白，用于正向選擇；僅在宿主菌 DB3.1 中可穩(wěn)定維持

2. attP1/attP2 位點(diǎn): Gateway 克隆系統(tǒng)重組位點(diǎn)

3. Pxyl/tetO 啟動子: 受脫水四環(huán)素誘導(dǎo)的反向選擇啟動子

4. secY 反義轉(zhuǎn)錄本: 脫水四環(huán)素誘導(dǎo)后表達(dá)，抑制金黃色葡萄球菌生長，用于反向選擇

5. TetR: 四環(huán)素阻遏蛋白編碼基因
   

6. 

7. 特性特征:

8. 溫度敏感性復(fù)制: 在 30-37°C 條件下可在金黃色葡萄球菌中復(fù)制；在 43°C 非允許溫度下復(fù)制被抑制，用于篩選同源重組整合事件

9. 兩步篩選機(jī)制:

   • 第一步 (整合)：43°C 培養(yǎng)選擇質(zhì)粒通過同源重組整合入染色體

   • 第二步 (切除)：脫水四環(huán)素誘導(dǎo) secY 反義 RNA 表達(dá)，抑制細(xì)胞生長，選擇質(zhì)粒切除和丟失事件

10. 高效性: 無需抗生素標(biāo)記即可高頻率獲得等位基因置換突變體

11. 應(yīng)用: 廣泛應(yīng)用于金黃色葡萄球菌的基因敲除、等位基因置換、點(diǎn)突變引入研究
    細(xì)菌培養(yǎng)條件:

• 大腸桿菌宿主菌：BioVector? DB3.1 感受態(tài)細(xì)胞 (用于 pKOR1 擴(kuò)增)

• 金黃色葡萄球菌宿主菌：BioVector? RN4220 或相應(yīng)臨床分離株

• 培養(yǎng)溫度：30-37°C (質(zhì)粒復(fù)制)；43°C (整合篩選)
  誘導(dǎo)條件: 脫水四環(huán)素 (終濃度根據(jù)實(shí)驗(yàn)優(yōu)化)
  質(zhì)控載體: BioVector? pUC19 質(zhì)粒
  宿主菌: BioVector? DB3.1 化學(xué)感受態(tài)細(xì)胞 (用于質(zhì)粒擴(kuò)增)
  質(zhì)量控制: 測序驗(yàn)證：100%正確；無菌檢測：陰性；酶切鑒定：符合預(yù)期條帶
  儲存與運(yùn)輸: -20°C 或干冰運(yùn)輸與保存
  僅供科研使用

English:
BioVector? pKOR1 Staphylococcus aureus Allelic Replacement Plasmid Datasheet

Product Name: BioVector? pKOR1 Plasmid
Synonyms: BioVector? pKOR1 Shuttle Vector
Chinese Name: BioVector? pKOR1 金黃色葡萄球菌等位基因置換質(zhì)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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