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The Single Step (KRX) Competent Cells: Efficient Cloning and
High Protein Yields
Jim Hartnett, M.S., Jill Gracyalny, B.S., and
Michael R. Slater, Ph.D.
Promega Corporation
We describe a new strain of Escherichia coli that has advantageous
features for cloning and screening plus engineered attributes for
tightly controlled protein expression. This new KRX strain is compatible
with blue/white screening and can be made highly competent. In addition,
KRX allows T7 RNA polymerase-based protein expression, one of the most
widely used expression systems due to its well-defined promoter and the
rapid elongation rate of the polymerase. In this strain, the T7 RNA
polymerase gene is controlled by a rhamnose promoter. When we used the
KRX strain to express firefly luciferase protein, the precise control of
the rhamnose operon resulted in a dramatic induction ratio of 1,700-fold
upon addition of rhamnose, whereas the leaky IPTG-inducible T7 RNA
polymerase-based system in BL21(DE3)-derived strains only showed an 8-
to 43-fold induction ratio, primarily due to high pre-induction levels
of protein expression. Protein expression levels in KRX for three
additional proteins were shown to be as high, or higher than, levels in
BL21(DE3)-derived strains.
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