Can I directly clone, propagate and express in BL21 without using TOP10?

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Answer

It is imperative that a cloning strain such as TOP10 be used for characterization of the plasmid, propagation, and maintenance. BL21 cells are wild-type for endA and recA, which could result in poor miniprep quality and a greater chance of plasmid rearrangements due to recombination. In addition, BL21 cells contain the T7 RNA polymerase gene which is expressed at low levels even in the absence of inducer. If the gene is toxic to E. coli, plasmid instability and/or cell death can result.

Answer Id: 3845

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What is the genotype of the BL21-AI™ competent cells?

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Answer

BL21-AI™: F- ompT hsdSB (rB-mB-) gal dcm araB::T7RNAP-tetA

The BL21-AI™ strain is an E. coli expression strain that is deficient in the lon protease and outer membrane protease, OmpT. The lack of these proteases reduces degradation of proteins expressed in this strain. The strain carries a chromosomal insertion of a cassette containing the T7 RNA polymerase and tetA genes in the araB locus, allowing expression of the two genes to be regulated by the araBAD promoter. The presence of the tetA gene confers resistance to tetracycline and permits verification of strain identity using tetracycline.

Answer Id: 3884

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Is the BL21-AI™ strain the best for expressing toxic proteins?

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The BL21-AI™ strain is suitable for high-level recombinant protein expression from any T7-based expression vector. Because T7 RNA polymerase levels can be tightly regulated, the BL21-AI strain is especially useful to express genes that may be toxic to other strains where basal expression of T7 RNA polymerase is leakier (e.g. BL21 Star™(DE3) or BL21(DE3)). The yield of recombinant protein obtained from BL21-AI is generally similar to that obtained from other BL21 strains.

Answer Id: 3885

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What is the evidence that my protein is toxic to bacteria when using the BL21-AI™ cells?

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Answer

You can assume the protein may be toxic when any of the following occurs:
1) No colonies are obtained after transformation and growth on plates, or a combination of large and small, irregular colonies appears on the plate.
2) The initial liquid culture does not grow.
3) It takes longer than 5 hours after a 1:20 dilution of the initial culture for the expression culture to reach an OD600=0.4.
4) The cells lyse after induction with L-arabinose (or L-arabinose and IPTG).

Answer Id: 3886

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