基因型

F- ompT hsdS(rB-mB-) gal dcm(DE3)pLysS Camr

产品说明

BL21(DE3)pLysS菌株携带pLysS质粒，具有氯霉素抗性。pLysS含有表达T7溶菌酶的基因，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，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。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)。BL21(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. BL21(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 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×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(0.1-2mM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. BL21(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。