



BL21(AI) Chemically Competent Cell 產(chǎn)品說明書
產(chǎn)品規(guī)格
BL21(AI): 10×100μl
pUC19(control vector):10pg/μl 10μl
保存條件:-80℃
基因型
F- ompT hsdSB(rB-mB-) gal dcm araB::T7RNAP-tetA
產(chǎn)品說明
BL21(AI)是大腸桿菌B/r型菌株(E.coli B/r)。BL21(AI) 來源于BL21菌株,為Lon蛋白酶和膜外蛋白酶OMPT的缺陷型菌株,這兩種酶的缺失有效防止異源蛋白在大腸桿菌體內(nèi)的降解。在培養(yǎng)基中添加L-阿拉伯糖可誘導(dǎo)araBAD啟動子下游T7RNA聚合酶的表達(dá)進(jìn)而促進(jìn)目的蛋白的表達(dá)。在培養(yǎng)基中添加葡萄糖可抑制araBAD啟動子下游T7RNA聚合酶的表達(dá)進(jìn)而抑制目的蛋白的表達(dá)。BL21(AI) 感受態(tài)細(xì)胞適用于任何以T7啟動子為基礎(chǔ)的表達(dá)載體,能夠進(jìn)行高水平的重組蛋白表達(dá)。因為菌株能夠?qū)w內(nèi)的T7 RNA聚合酶水平進(jìn)行高效調(diào)節(jié),BL21(AI) 感受態(tài)細(xì)胞能夠表達(dá)對其他BL21細(xì)胞有毒性或抑制生長的蛋白。普通重組蛋白在BL21(AI) 菌株中獲得產(chǎn)量和其他BL21菌株產(chǎn)量相當(dāng);對大部分毒性蛋白,在BL21(AI) 菌株中獲得的產(chǎn)量高于BL21(DE3)pLysS菌株或BL21(DE3)菌株。BL21(AI) 感受態(tài)細(xì)胞由特殊工藝制作,pUC19質(zhì)粒檢測轉(zhuǎn)化效率高達(dá)108cfu/μg DNA。
操作方法
1. BL21(AI)感受態(tài)細(xì)胞從-80℃拿出,迅速插入冰中,5分鐘后待菌塊融化,加入目的DNA(質(zhì)?;蜻B接產(chǎn)物)并用手撥打EP管底輕輕混勻(避免用槍吸打),冰中靜置25分鐘。
2. 42℃水浴熱激45秒,迅速放回冰上并靜置2分鐘,晃動會降低轉(zhuǎn)化效率。
3. 向離心管中加入700μl不含抗生素的無菌培養(yǎng)基(2YT或LB),混勻后37℃,200rpm復(fù)蘇60分鐘。
4. 5000rpm離心一分鐘收菌,留取100μl左右上清輕輕吹打重懸菌塊并涂布到含抗生素的2YT 或LB培養(yǎng)基上。
5. 將平板倒置放于37℃培養(yǎng)箱過夜培養(yǎng)。
注意:
1. Brian Caliendo (Voigt 實驗室)報道過pCP20質(zhì)粒比較難于轉(zhuǎn)化到這個感受態(tài)細(xì)胞中,而pCP20轉(zhuǎn)化到其他菌株中都很正常,但是菌體原因未知。
2. 不加葡萄糖,BL21(AI) 細(xì)胞的araBAD啟動子下游的本底蛋白表達(dá)水平仍然很低,加入葡萄糖后能夠進(jìn)一步的降低本底蛋白的表達(dá)水平。
Sample Induction Protocol (for reference only)
1. Pick 3-4 transformants for overnight culture in 5 mL LB medium containing antibiotic to select for your expression plasmid. Grow overnight at 37°C with shaking until the OD600 reaches 0.6-1.0.
2.Use the overnight cultures to inoculate fresh LB medium containing antibiotic to an OD600 of 0.05-0.1 (~1:20 dilution of the overnight culture). This dilution allows the cells to quickly return to logarithmic growth and reach the appropriate cell density. Use a volume appropriate for taking time points, if desired.
3.Use the remainder of each overnight culture to create glycerol stocks. Once you have identified the clone that best expresses your protein, you can use the glycerol stock to perform additional expression experiments.
4.Grow the cultures until they reach mid-log phase (OD600 ~0.4; 2 to 3 hours).
5.Induce the cultures (see below), and culture for an additional 2-3 hours. You may also take time points to analyze for optimal expression of your protein.
For T7 expression vector containing the lacI gene (e.g. Invitrogen’s pET vectors), induce by adding L-arabinose to a final concentration of 0.2% AND IPTG to a final concentration of 1 mM.
For T7 expression vector with no lacI gene (e.g. Invitrogen’s pCR?T7 vectors), induce by adding L-arabinose to a final concentration of 0.2%. Culture for an additional 2-3 hours.
6.Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000×g for 10 minutes at 4℃.
7. 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 / thiogalactopyranoside) by dissolving 2.38 g of IPTG in dd water and adjust the final volume to 10 ml. Filter sterilize before use.
注意事項
1. 感受態(tài)細(xì)胞最好在冰中緩慢融化,插入冰中8分鐘內(nèi)加入目標(biāo)DNA,不可在冰中放置時間過長,長時間存放會降低轉(zhuǎn)化效率。
2. 混入質(zhì)粒時應(yīng)輕柔操作。
3. 轉(zhuǎn)化高濃度的質(zhì)??上鄳?yīng)減少最終用于涂板的菌量。
4. 誘導(dǎo)時,IPTG濃度可選(0.1-2mM均可)。
5. 為獲得需要量的蛋白,最佳誘導(dǎo)時間,溫度,IPTG濃度需實驗者優(yōu)化。
建議選擇該菌株進(jìn)行蛋白表達(dá)的條件如下:
1. 使用T7啟動子載體(高拷貝或者低拷貝都可以)進(jìn)行蛋白表達(dá)。
2. 使用其他BL21菌株進(jìn)行蛋白表達(dá)時,觀察到明顯的細(xì)菌生長的抑制作用。
3. 表達(dá)一個已知的毒性蛋白。