产品规格

基因型

产品说明

Rosetta-gami(DE3)pLysS 菌株聚合了不同原核表达菌株的优势：

• Rosetta-gami赋予其 Rosetta和Origami的优点——补充大肠杆菌缺乏的6种稀有密码子(AUA, AGG, AGA, CUA, CCC, GGA)对应的tRNA，提高外源基因的表达水平，并且包含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，其突变有利于高效形成正确折叠的含有二硫键的蛋白，增强蛋白的可溶性。
• 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)适合T7启动子诱导的蛋白表达。
• 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。Rosetta-gami(DE3)pLysS 菌株具有卡那霉素，氯霉素，链霉素，四环素抗性，为亮氨酸生长缺陷型菌株。BFB生命科学的Rosetta-gami(DE3)pLysS感受态细胞由特殊工艺制作，pUC19质粒检测转化效率高达10⁸ cfu/μg DNA。

操作方法

1. Rosetta-gami(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DNA（质粒或连接产物）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34 µg/ml氯霉素及所选质粒筛选抗生素的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.
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
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
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. 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（0.1-2 mM均可）。
5. 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。
6. Rosetta-gami(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。
7. 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。