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1、常用大肠杆菌基础信息及使用说明菌株目录BL21 (2)BL21(DE3) (4)BL21(DE3)pLysS (6)BL21 Star(DE3) (8)BL21(AI) (10)OverExpress C43(DE3) (13)M15(pREP4) (15)CopyCutter EPI400 (16)Rosetta(DE3) (18)XL10 (20)Mach1-T1 (22)DH5a (23)TOP10 (24)BL21基因型F- dcm omp T hsd S(r B- m B-) gal产品说明作为E.coli B宿主菌，主要用来进行蛋白表达，细胞内缺少Ion蛋 白酶和 ompT 外膜蛋
2、白酶，能够有效避免目的蛋白的降解；当使用 CE6噬菌体启动子时，BL21感受态细胞对目的蛋白在未诱导情况下的 蛋白表达严密控制；用于非T7启动子驱动的基因表达，表达水平极高。操作方法1. BL21感受态细胞从-80弋拿出，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弋水浴热激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川不含抗生素的无菌培养基(2YT或LB), 混匀后37C, 200rpm复苏60分钟。4.5000rpm离心一分钟收菌，留取100川左右上清轻轻
3、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37C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 37Covernight.
4、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 37Cuntil the OD 600 reaches 0.5-0.8.5. (Optional)Pipet 1ml of the cul
5、tures into clean microcentrifuge tubes and place the tubes on ice until needed for gel analysis or storage at -20C. These will serve as the noninducedcontrol 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
6、the protein.7. Incubate with shaking at 120 rpm at 37Cfor 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 cen
7、trifugatio n at 5,000xg for 10 min at 4C.9. Remove the supernatant and store the cell pellet at - 20C(storage at lower temperatures is also acceptable).IPTGPrepare a 1 M solution of IPTG (Isopropyl-B-D- thiogalactoside;Isopropyl-D-thiogalactopyra noside)bydissolving 2.38 g of IPTG in dd water and ad
8、just the final volume to 10 ml. Filter sterilize before use.BL21(DE3)基因型F- omp T hsd S B(r B- m B- ) gal dcm(DE3)产品说明BL21(DE3)菌株用于高效表达克隆于含有噬菌体T7启动子的表达 载体(如pET系列)的基因。入噬菌体DE3区含有T7噬菌体RNA聚合 酶，该区整合于BL21的染色体上，所以称为BL21(DE3)。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。BL21(DE3)感受态 细胞由特殊工艺制作，PUC19
9、质粒检测转化效率达107cfu/|jg DNA。操作方法1. BL21(DE3)感受态细胞从-80C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。2.42C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川不含抗生素的无菌培养基(2YT或LB), 混匀后37C, 200rpm复苏60分钟。4.5000rpm离心一分钟收菌，留取100pl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37C培养箱过夜培养。Sample
10、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 37Covernight.3. Inoculate 50 ml of LB medium containing the appropriat
11、e 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 37Cuntil the OD 600 reaches 0.5-0.8.5. (Optional)Pipet 1ml of the cultures into clean microcentrifuge tubes and place the tube
12、s on ice until needed for gel analysis or storage at -20C. These will serve as the noninduced 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 37Cfo
13、r 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 cen trifugatio n at 5,000xg for 10 min at 4C.9. Remove the s
14、upernatant and store the cell pellet at - 20C(storage at lower temperatures is also acceptable).IPTGPrepare a 1 M solution of IPTG (Isopropyl-B-D- thiogalactoside;Isopropyl-D-thiogalactopyra noside)bydissolving 2.38 g of IPTG in dd water and adjust the final volume to 10 ml. Filter sterilize before
15、use.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。BL21(DE3)pLysS基因型F-omp T hsd S(r B-m B-) gal dcm(DE3)pLysS Cam r 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
16、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 整合了入噬菌体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/ug DNA。操作方法1. BL21(DE3)pLysS感受态细胞从-80C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. 42C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川不含抗生素的无菌
