OverExpress C43(DE3)化学表达感受态细胞

OverExpress C43(DE3)化学表达感受态细胞

OverExpress C43(DE3) Chemically Competent Cell

OVEREXPRESS C43(DE3)感受态细胞基因型：

F–ompT hsdSB (rB- mB-) gal dcm (DE3)

OVEREXPRESS C43(DE3)感受态细胞说明：

OverExpress C43(DE3)、OverExpress C41(DE3)两个菌株均起源于BL21(DE3)，其优点是可以高效表达毒性蛋白或疏水性蛋白。OverExpress C41(DE3)跟BL21(DE3)的区别在于其基因组含有至少一个未知突变，这个未知突变使其获得了高效表达毒性蛋白 (1-5)的能力，此突变位点参与大肠杆菌表达毒性蛋白时的细胞死亡途径；OverExpress C43(DE3)来源于OverExpress C41(DE3)，是通过筛选OverExpress C41(DE3)对另一个不同毒性蛋白的抗性菌株获得。OverExpress C43(DE3)菌株具有比OverExpress C41(DE3)更强的表达毒性蛋白和疏水性蛋白的能力，所以说OverExpress C43(DE3) 菌株与BL21(DE3)相比拥有至少两个未知突变，正是这两个未知突变使其获得了更广泛的表达毒性蛋白或疏水性蛋白的能力。此菌株含有DE3区，可同时表达T7 RNA聚合酶和大肠杆菌RNA聚合酶，可用于pET系列，pGEX，pMAL等质粒的蛋白表达。OverExpress C43(DE3)感受态细胞由特殊工艺制作，pUC19质粒检测转化效率可达5×10⁸ cfu/μg DNA。  

OVEREXPRESS C43(DE3)感受态细胞操作方法：

1. OverExpress C43(DE3)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℃，200 rpm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相应抗生  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.

OVEREXPRESS C43(DE3)感受态细胞注意事项：

1. 感受态细胞最好在冰上融化。

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.

OVEREXPRESS C43(DE3)感受态细胞保存条件： -80℃

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