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pBT2

價格:4500元

聯系方式:I47-825O-882O

相關技術服務:質粒構建    基因合成    質粒大提

買家導航

載體基本信息

出品公司: Bruckner R實驗室
載體名稱: pBT2
質粒類型: 金黃色葡萄球菌基因敲除載體,金葡菌基因敲除載體
高拷貝/低拷貝: 低拷貝
克隆方法: 限制性內切酶,多克隆位點
啟動子: --
載體大小: 6.97kb
5' 測序引物及序列: --
3' 測序引物及序列: --
載體標簽: --
載體抗性: 氨芐青霉素
篩選標記: 氯霉素
克隆菌株: DH5α 等
宿主細胞(系): 金黃色葡萄球菌等革蘭氏陽性菌
備注: 該載體是大腸桿菌-金黃色葡萄球菌穿梭載體,低拷貝,在大腸中抗性為氨芐,在金葡菌中抗性為氯霉素。該質粒具有溫度敏感性。

生物風對金黃色葡萄球菌的基因敲除具有豐富的實驗經驗,并且成功敲除過很多金黃色葡萄球菌基因,請有需要對金黃色葡萄球菌進行基因操作技術服務的老師和我們聯系。

參考文獻:Bruckner R: Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS microbiology letters 1997, 151(1):1-8.

相關的配套菌株為RN4220金黃色葡萄球菌, 相關的載體為pKOR1載體。
產品目錄號: --
穩定性: 穩表達
組成型/誘導型: 組成型
病毒/非病毒: 非病毒

載體質粒圖譜和多克隆位點信息

pBT2載體圖譜

載體簡介

Protocols for gene deletion in Staphylococcus aureus   Nov. 1, 2007


Preparation of competent Staphylococcus aureus cells

  1. 1. Remove Staphylococcus aureus cells from the vial with a sterile toothpick or inoculation loop, and streak it out on LB agar.
  2. 2. Incubate at 37°C overnight.
  3. 3. Pick a single colony and inoculate it in 5-10 ml of LB. Grow at 37°C overnight.
  4. 4. Add 1 ml overnight culture to 100 ml LB medium in a 500 ml flask, and shake at 37°C until an OD600 of 0.4 is reached (approximately 90–120 min).
  5. 5. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom centrifuge tube.
  6. 6. Collect the cells by centrifugation at low speed (5-10 min, 2500 x g, 4°C).
  7. 7. Discard the supernatant carefully. Always keep the cells on ice.
  8. 8. Resuspend the cells gently in 0.5 M sucrose (10-15 ml for a 100 ml culture) at 4°C and keep the suspension on ice for additional 5 min.
  9. 9. Collect the cells by centrifugation (5 min, 2500 x g, 4°C).
  10. 10. Discard the supernatant carefully. Repeat step 8 and 9.
  11. 11. Resuspend the cells carefully in 1 ml ice-cold 0.5 M sucrose and keep the suspension on ice for 15 min.
  12. 12. Prepare aliquots of 100–200 μl in sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at –70°C.

Construction of deletion vector
  1. 1. PCR amplify a 400 bp fragment upstream and a 400 bp fragment downstream of the target gene.
  2. 2. PCR amplify the ermB (Em resistance marker) from pECI.
  3. 3. Digest the three fragments, ligate, and PCR amplify the ligated product.
  4. 4. Purify the PCR product, double digest it, and ligate it into pBT2.
  5. 5. Transform the ligated product into E. coli.
  6. 6. Pick clones that can grow on the LB plate containing Em (100 mg/ml), purify the plasmid and digest it.
  7. 7. If the result of enzyme digestion is correct, get further confirmation by sequencing.  

Procedure for electroporation
  1. 1. Mix 500 ng of plasmid DNA with electrocompetent Staphylococcus aureus cells and place them in a Gene Pulser cuvette with a 0.2 cm electrode gap.
  2. 2. The settings for electroporation are as follows: Voltage, 2.5 kV; capacitor, 50 μF; resistance, 200 ohms.
  3. 3. After electroporation the cells are immediately placed in 400 μl of TSB with shaking (200-220 rpm, 37°C) for 1h. Plate the cells on Em-containing medium and incubate at 37°C.

Modify deletion vector
  1. 1. Before transform the plasmid into Staphylococcus aureus NCTC8325, the plasmid should be transformed into Staphylococcus aureus RN4220.
  2. 2. Pick clones, after overnight growth, extract the plasmid.
  3. 3. Then the plasmid is modified and can’t be digested by restriction enzyme system of NCTC8325.

Deletion of target gene
  1. 1. Extract plasmid from RN4220, transform it into NCTC8325.
  2. 2. Pick up clones, incubate in B-medium, 30°C, grow to late-stationary phase, then change temperature to 40°C, and grow overnight.
  3. 3. 1: 100 dilute the culture into fresh B-medium, and grow overnight.
  4. 4. Follow step 3, spread 1 μl overnight culture (diluted into 100 μl) on agar plate (containing Em 2.5 mg/ml). Screen clones which are Em-resistant and Cm-sensitive.
  5. 5. Repeat step 4 until Em-resistant, Cm-sensitive clones are found.
  6. 6. Extract genome DNA of these clones, use PCR for further check.


生物風對金黃色葡萄球菌的基因敲除具有豐富的實驗經驗,并且成功敲除過很多金黃色葡萄球菌基因,請有需要對金黃色葡萄球菌進行基因操作技術服務的老師和我們聯系。

載體序列

載體質粒圖譜pdf版和相關資料下載

載體應用舉例

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