OrigamiB(DE3)pLysS Chemically Competent Cell说明书

产品货号： ML-G27027                     

保存条件： -80℃   

产品规格:  10×100μl  50×100μl

产品介绍

基因型                 

F-ompT hsdSB(rB-mB-) gal dcm lacY1 ahpC (DE3) gor522::Tn10 trxB (KanR,TetR) pLysS CamR 

简要说明

Origami B (DE3) pLysS菌株携带pLysS质粒，具有氯霉素抗性。pLysS可表达T7溶菌酶（T7溶菌酶可以作用于大肠杆菌细胞壁上的肽聚糖溶解大肠杆菌，还可与T7 RNA聚合酶结合抑制其转录活性，进而降低目的基因的背景表达水平，但不干扰IPTG诱导的表达），适合表达毒性蛋白和非毒性蛋白。

Origami B (DE3) pLysS菌株表达突变的硫氧还蛋白还原酶 (thioredoxin reductase) (trxB)和谷胱甘肽还原酶 (glutathione reductase) (gor)，这两个酶是还原途径的关键酶，突变后有利于形成正确折叠的含有二硫键的蛋白，增强蛋白的可溶性。

此外，该菌株染色体整合了λ噬菌体DE3区 (DE3区含有T7噬菌体RNA聚合酶)，可同时表达T7 RNA聚合酶和大肠杆菌RNA聚合酶，可用于pET系列，pGEX，pMAL等质粒的蛋白表达，具有氯霉素、卡那霉素和四环素抗性，不能用于具有氯霉素，卡那霉素抗性质粒的表达。Origami B (DE3) pLysS感受态细胞由特殊工艺制作，pUC19质粒检测转化效率达108 cfu/μg DNA。

操作说明

1. 取100μl冰上融化的Origami B (DE3) pLysS感受态细胞，加入目的质粒并轻轻混匀，冰上静置25分钟。

2. 42℃水浴热激45秒，迅速放回冰上并静置2分钟，晃动会降低转化效率。

3. 向离心管中加入700μl不含抗生素的无菌培养基(2YT或LB)，混匀后37℃，200rpm复苏60分钟。

4. 5000rpm离心一分钟收菌，留取100μl左右上清轻轻吹打重悬菌块并涂布到含相应抗生素的2YT或LB培养基上。

5. 将平板倒置放于37℃培养箱过夜培养。

注意事项

1. MLBio感受态细胞*在冰上融化。

2. 混入质粒时应轻柔操作。

3. 转化高浓度的质粒可相应减少最终用于涂板的菌量。

4. 诱导时，IPTG浓度可选(0.1-2mM均可)。

5. 为获得需要量的蛋白，*诱导时间，温度，IPTG浓度需实验者优化。

6. 具有卡那霉素抗性，不能用于具有卡那霉素抗性质粒的表达。

Sample Induction Protocol (for reference only)

1. Inoculate a single colony from a freshly streaked plate into 5 ml of LB medium containing the appropriate antibiotic for the plasmid and host strain.

2. Incubate with shaking at 200 rpm at 37℃ overnight. 

3. Inoculate 50 ml of LB medium containing the appropriate antibiotic with 0.5 ml of the overnight culture prepared in step 2(use the 500 ml triangular flask as the container would be better).

4. Incubate with shaking at 150 rpm at 37℃ until the OD 600 reaches 0.5-0.8.

5. (Optional)Pipet 1ml of  the cultures into clean microcentrifuge tubes and  place the tubes on ice until needed  for gel analysis or storage at  -20℃. These will serve as the non-induced control samples.     

6. Add IPTG to a final concentration of 1 mM.  Optimal time for induction of the target protein may vary from 2-16 hours, depending on the protein.

7. Incubate with shaking at 120 rpm at 37℃ for 3-4 hours. To determine the optimal time for induction of the target protein, it is recommended that a time course experiment be performed varying the induction from 2-16 hours.

8. Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000 x g for 10

minutes at 4℃.

9. Remove the supernatant and store the cell pellet at -20℃ (storage at lower temperatures is also acceptable). 

IPTG

Prepare a 1 M solution of IPTG (Isopropyl-β-D-thiogalactoside; Isopropyl-β-D-thiogalactopyranoside) bydissolving 2.38 g of IPTG in dd water and adjust the final volume to 10 ml. Filter sterilize before use.