BL21(DE3) problems

Kresten kresten at my-deja.com
Sun Nov 28 10:41:05 EST 1999


From:
http://www.novagen.com/html/inno/n9d.html


Addition of glucose to growth media to
reduce background expression in DE3 host
strains.

Robert Novy — Novagen, Inc.

pET expression host strains are DE3 lysogens in which the gene for T7
RNA polymerase is controlled by the lacUV5 promoter. In contrast to the
wildtype lac promoter, the lacUV5 promoter is relatively insensitive to
cAMP/CRP mediated stimulation (1). However, more recently it has been
demonstrated that when DE3 hosts are grown to stationary phase in media
lacking glucose, cAMP mediated derepression of both the wildtype and
lacUV5 promoters occurs (2). The resulting expression of T7 RNA
polymerase can lead to instability of pET plasmids.

Although growing DE3 hosts containing pET recombinants to s tationary
phase is never recommended, derepression can be effectively avoided in
overnight (16 h) cultures by adding glucose to a final concentration of
1% in the growth media. Glucose inhibits cAMP production
thereby avoiding the derepression phenomenon. Typically, glucose
addition is not required to maintain plasmid stability; however, in
cases of extremely toxic target genes the addition of glucose to
both agar plates and liquid media should help further decrease the
already low levels of basal expression.

References

1.Eron, L. and Block, R. (1971) Proc. Natl. Acad. Sci. USA 68,
1828–1832.
2.Grossman, T.H., Kawasaki, E.S., Punreddy, S.R. and Osburne, M.S.
(1998) Gene 209, 95–103.



In article <0.58fd35e5.25717a47 at aol.com>,
  DOCTORHIM at AOL.COM wrote:
> Glucose is irrelevant since  the LacUV5 promoter is used for T7 RNA
> polymerase.  UV5 is not catabolite repressible.
>

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Kresten Lindorff Larsen, Dept. Yeast Genetics


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