One Shot™ BL21(DE3)pLysE 化学感受态大肠杆菌
One Shot&trade; BL21(DE3)pLysE 化学感受态<i>大肠杆菌</i>
Invitrogen™

One Shot™ BL21(DE3)pLysE 化学感受态大肠杆菌

One Shot BL21(DE3)pLysE Chemically Competent E. coli cells are ideal for use with bacteriophage T7 promoter-based expression systems such as了解更多信息
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货号数量
C65650320 x 50μL
货号 C656503
价格(CNY)
3,202.00
Each
添加至购物车
数量:
20 x 50μL
价格(CNY)
3,202.00
Each
添加至购物车

One Shot BL21(DE3)pLysE Chemically Competent E. coli cells are ideal for use with bacteriophage T7 promoter-based expression systems such as pRSET and pET. The pLysE plasmid carried by the BL21(DE3)pLysE strain produces higher amounts of T7 lysozyme than pLysS to further reduce basal level expression of the gene of interest. Like pLysS, pLysE confers resistance to chloramphenicol (CamR) and contains the p15A origin for compatibility with plasmids containing the ColE1 or pMB1 origin (i.e. pUC- or pBR322- derived plasmids). Recombinant proteins that are nontoxic to E. coli are generally expressed at higher levels in BL21(DE3) cells as compared to the BL21(DE3)pLysE strain with tighter expression control. The BL21(DE3)pLysE strain is recommended for use when expressing toxic genes (alternatively, BL21-AI could be considered).

The BL21 (DE3) E. coli strain is one of the most popular host strains to produce recombinant proteins and adapted in research or industrial applications (e.g., first biopharmaceutical recombinant human insulin). This strain had been engineered to carry the λDE3 lysogen that contains the T7 RNA polymerase gene under control of the lacUV5 promoter. IPTG is required to induce expression of the T7 RNA polymerase cascade system that in turn transcribe the T7 promoter regulated target gene.

BL21(DE3) is a derivative of the E. coli B strain that does not contain the lon protease and is also deficient in the outer membrane protease OmpT. The lack of two key proteases reduces degradation of heterologous proteins expressed in the strains. Additionally, several other strain features make them especially suited for protein production, namely fast growth in minimal medium, a lower acetate production when grown in high glucose media, and the ability to reach high cell density.

One Shot BL21(DE3)pLysE Chemically Competent E. coli offer:
• Transformation efficiency of >1 x 107 cfu/μg control plasmid DNA
• Ion and OmpT protease deficiency reduces degradation of recombinant protein
• IPTG-inducible lacUV5 promoter controls expression of the T7 RNA polymerase
• pLysE (CamR) plasmid for reduced basal expression in uninduced cells
hsdS mutation allows efficient transformation of unmethylated DNA
• Fast growth in minimal medium and ability to reach high cell density

BL21(DE3)pLysE E. coli are ideal for use with T7 promoter-based expression vectors. Like BL21(DE3)pLysS E. coli, BL21(DE3)pLysE cells carry the lambda DE3 lysogen. In addition, BL21(DE3)pLysE cells contain the pLysE plasmid, which constitutively expresses T7 lysozyme. T7 lysozyme reduces the basal expression of target genes by inhibiting T7 RNA polymerase. The pLysE plasmid in BL21(DE3)pLysE expresses higher levels of T7 lysozyme than the pLysS plasmid in BL21(DE3)pLysS. The BL21(DE3)pLysE strain provides tighter control of T7 RNA polymerase, which is necessary when the recombinant protein to be expressed is toxic.

Easy-to-use One Shot format
 
BL21(DE3)pLysE Chemically Competent E. coli cells are supplied in the convenient, single-reaction One Shot format. The single-tube, single-use format allows all steps of the transformation protocol, up to plating, to take place in the same tube, thereby helping save time and prevent contamination.

Genotype
FompT hsdSB (rB–, mB–) gal dcm (DE3) pLysE(CamR)

Find the strain and format that you need
We offer other E. coli strains for protein expression.
For expression of toxic proteins, consider BL21-AI One Shot Chemically Competent E. coli.
BL21 Star (DE3) and other strains are available in MultiShot formats for high throughput applications.
Explore bacterial growth media formats.
We offer a variety of systems for the expression of recombinant proteins in E. coli, the Champion pET expression system provides the highest level of protein production available in any expression system.

仅供科研使用。不可用于诊断程序。
规格
耐抗生素细菌Yes (Chloramphenicol)
蓝色/白色筛查
是否可克隆甲基化 DNA
是否含 F' 附加体缺乏 F’附加体
高通量能力不兼容高通量应用(手动)
提高质粒质量
提高蛋白稳定性Yes (lon, ompT)
提高 RNA 稳定性Yes (pLysE)
制备无甲基化 DNAYes (dcm)
产品线One Shot™
产品类型感受态细胞
数量20 x 50μL
减少克隆重组现象
运输条件Dry Ice
T1 噬菌体 - 抗性 (tonA)
有毒蛋白质No
转化效率级别亚克隆效率 (10^6-10^7 cfu⁄µg)
产品规格One Shot
促进剂T7
种属大肠杆菌
Unit SizeEach
内容与储存
• BL21 Star (DE3)pLysE Chemically Competent E. coli (21 x 50 μL); store at –80°C
• pUC19 DNA (1 x 50 μL); store at –20°C
• S.O.C. Medium (6 mL); store at 4°C or room temperature

常见问题解答 (FAQ)

我的目的基因对细菌细胞有毒性作用。可采取哪些预防措施?

有多种措施可防止由毒性基因本底水平表达带来的问题。这些方法均基于T7或Champion表达质粒的设计和构建是正确的:

•在不含T7 RNA聚合酶(即,DH5α)的菌株中繁殖和维持表达质粒。
•如果使用BL21 (DE3)菌株,应在室温而非37°C下生长24-48小时。
•新鲜转化严格调控的大肠杆菌菌株,如BL21-AI菌株。
•转化实验后,将转化反应接种在含100 μg/mL氨苄青霉素和0.1%葡萄糖的LB板上。葡萄糖可抑制T7 RNA聚合酶的本底表达。
•完成BL21-AI菌株的转化后,挑选3或4个转化株直接接种到新制备的含100 μg/mL 氨苄青霉素或50 μg/mL 羧苄青霉素(以及0.1%葡萄糖,如果需要的话)的LB培养基中。当培养物的OD600值达到0.4时,可加入终浓度为0.2%的L-阿拉伯糖来诱导重组蛋白的表达。
•在表达实验中,在生长培养基中补充0.1%葡萄糖和0.2%阿拉伯糖。
•尝试使用调控型细菌表达系统,如我们的pBAD系统。

如何能够知道蛋白质发生降解或者存在密码子使用偏好?

通常,如果您看见1-2条主带,则表示密码子使用偏好导致了翻译过早终止。发生降解时,通常可看到梯度条带。发生降解时,可尝试在裂解缓冲液中加入蛋白酶抑制剂有助于防止降解。如果是这种情况,可通过时间点实验来确定收细胞的最佳时间。

我得到了包涵体,该怎么办?

如果存在溶解性问题,可尝试在诱导时降低温度或减少IPTG用量。也可尝试使用不同的、更严格的菌株进行表达。此外,在表达期间,也可将细菌培养基的葡萄糖含量增加至1%。

我的蛋白得率很低,应该如何进行改善?

使用新鲜的细菌培养物进行接种,因为新鲜的细菌菌落通常可得到较高的蛋白得率。

检查重组蛋白序列中的不常用密码子。利用常用密码子代替稀有密码子,可显著提高表达水平。例如,精氨酸密码子AGG和AGA是大肠杆菌的不常用的密码子,所以这些密码子的tRNA水平低。

进行蛋白纯化时,在缓冲液中加入蛋白酶抑制剂,如PMSF。使用新鲜配制的PMSF,因为PMSF被稀释到水溶液中后30分钟内会失效。

如果您在表达实验中使用氨苄青霉素进行筛选,则可能会因为筛选条件缺失而出现质粒不稳定。这是因为氨苄青霉素被β-内酰胺酶破坏,或者在由细菌代谢物造成的酸性培养基条件下发生水解。在转化和表达实验中,可使用羧苄青霉素替代氨苄青霉素。

重组蛋白可能对细菌细胞具有毒性作用。应选择更严格调控的系统用于表达,如BL21-AI。也可考虑尝试使用不同的表达系统,如pBAD系统。

我的细胞生长非常缓慢,也没有得到任何蛋白表达。我该如何解决这个问题?

当您的目的基因具有毒性时这种情况很常见。尝试使用更严格调控的系统,如BL21 (DE3) (pLysS)或BL21 (DE3) (pLysE)或BL21(AI)。