基因型

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

產(chǎn)品說明

BL21(DE3)pLysS菌株攜帶pLysS質(zhì)粒，具有氯霉素抗性。pLysS含有表達T7溶菌酶的基因，T7溶菌酶可以作用于大腸桿菌細胞壁上的肽聚糖溶解大腸桿菌，能夠降低目的基因的背景表達水平，但不干擾IPTG誘導(dǎo)的表達，適合表達毒性蛋白和非毒性蛋白。該菌株染色體整合了λ噬菌體DE3區(qū)(DE3區(qū)含有T7噬菌體RNA聚合酶)。BL21(DE3)pLysS 感受態(tài)細胞由特殊工藝制作，pUC19質(zhì)粒檢測轉(zhuǎn)化效率高達108 cfu/μg DNA。 

操作方法

1. BL21(DE3)pLysS感受態(tài)細胞從-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)粒或連接產(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34 μg/ml氯霉素及所選質(zhì)粒篩選抗生素的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)。

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.

注意事項

1. 感受態(tài)細胞最好在冰中緩慢融化，插入冰中8分鐘內(nèi)加入目標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。

6. BL21(DE3)pLysS 菌株攜帶 pLysS質(zhì)粒，除復(fù)蘇培養(yǎng)基為無抗生素外，其余所用培養(yǎng)基、培養(yǎng)液均應(yīng)含有34 μg/ml氯霉素，以防質(zhì)粒丟失。