General description
Sigma's BL21-T1R competent E. coli cells are grown and made chemically competent using an optimized procedure specific to the strain, followed by strain verification and efficiency testing. The cells are provided in frozen 50 μl aliquots for convenience. Each aliquot can be used for a single transformation.
BL21(DE3)pLysE-T1R competent Escherichia coli is an expression strain suitable for high level induction and expression of genes from any T7 promoter-based expression vector. BL21(DE3)pLysE-T1R is a B/r strain bacteria which naturally lacks the lon protease and is also deficient in the outer membrane protease ompT. The absence of proteolytic activity from these two proteases may reduce degradation of some heterologous proteins expressed in the strain. The DE3 designation indicates the strain is lysogenic for a lambda prophage containing an inducible T7 RNA polymerase, which is under the control of the lacUV5 promoter. T7 RNA polymerase expression is induced by addition of 1 mM IPTG to the culture. The pLysE plasmid expresses T7 lysozyme, a natural inhibitor of the T7 polymerase, allowing for improved transcriptional control and reduction of "leaky" expression. The pLysE plasmid expresses T7 lysozyme at higher levels than the pLysS plasmid, conferring a greater level of control over the T7 polymerase. This is usually only required when the recombinant protein to be expressed may be toxic to the cell. The pLysE plasmid also renders the cell resistant to chloramphenicol (CmR) (5 μg/ml in liquid media and 25 μg/ml on plates) and contains the p15A origin. The p15A origin allows pLysE to be compatible with plasmids containing the ColE1 or pMB1 (derivative of pBR322) origin. In addition, the tonA genotype confers resistance to the lytic bacteriophages T1 and T5 for protection of clonal stocks.
The cells have a transformation efficiency of >1x106 cfu/μg when transformed with non-saturating amounts of pUC19 plasmid DNA.
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