• Origami2(DE3)pLysS Chemically Competent Cell +EC1023.pdf

产品规格 （CAT#: EC1023）

基因型

Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC galE galK rpsL F′[lac⁺ lacI^q pro] (DE3) gor522::Tn10 trxB pLysS (Cam^R, Str^R, Tet^R) 

产品说明

      Origami2(DE3)pLysS菌株是K-12的衍生菌株，含有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)基因，它们是还原途径的两个关键酶，其突变有利于含有二硫键蛋白的正确折叠，增强蛋白的可溶性；同时该菌株不具有卡那霉素抗性，可用于具有卡那霉素抗性质粒的蛋白表达。Origami2(DE3)pLysS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)，该区整合于大肠杆菌的染色体上，可同时表达T7 RNA聚合酶和大肠杆菌RNA聚合酶，用于pET系列，pGEX，pMAL等质粒的蛋白表达，该菌株携带的pLysS质粒含有表达T7溶菌酶的基因，能够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。Origami2(DE3)pLysS菌株具有氯霉素，链霉素，四环素抗性，为亮氨酸生长缺陷型菌株。Origami2(DE3)pLysS感受态细胞由特殊工艺制作，pUC19质粒检测转化效率约10⁶ cfu/μg DNA。

操作方法

1.Origami2(DE3)pLysS感受态细胞从-80℃拿出，迅速插入冰中，5分钟后待菌块融化，加入目的质粒并用手拨打EP管底轻轻混匀(避免用枪吸打)，冰中静置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700 μl不含抗生素的无菌培养基 (2YT或LB)，混匀后37℃，200 rpm复苏60分钟。

4.5000 rpm离心一分钟收菌，留取100 μl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。

Sample Induction Protocol (for reference only)

1.Inoculate a single colony from a freshly streaked plate into 3ml 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. (0.6 recommended; about 2.5h).

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 2-4 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×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配制

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

氯霉素配制

Chloramphenicol  34 mg/ml in ethanol. Store at -20℃. Use at 34 µg/ml.

注意事项

1.感受态细胞最好在冰中缓慢融化，插入冰中8分钟内加入目标DNA，不可在冰中放置时间过长，长时间存放会降低转化效率。

2. 转化高浓度的质粒可相应减少最终用于涂板的菌量。

3.诱导时，IPTG浓度可选（0.1-2 mM均可）。

4.为获得需要量的蛋白，最佳诱导时间，温度，IPTG浓度需实验者优化。

5.Origami2(DE3)pLysS菌株携带  pLysS质粒，除复苏培养基为无抗生素外，其余所用培养基、培养液均应含有 34 µg/ml 氯霉素，以防质粒丢失。