semi toxic

David Alexander dc.alexander at utoronto.ca
Thu May 10 14:06:12 EST 2001


Hello All,

I'm scratching my head over a recent transformation.
In short, I ligated a insert containing my gene of interest, in frame, into a pET vector
transformed into E. coli DH5a, and plated on LB plates (+ antibiotics).

Transformants appeared, but it took 3 days, and even after a week, the colonies were really tiny.
(the vector-only control grew fine, luscious colonies after 24 hours)
I tried scraping some of the tiny colonies into a tube of LB broth (no antibiotics),
but one week later, there is just the faintest turbidity.

I realize that pET vectors are leaky, and when trying to express a protein, toxicity is an issue.
However,  I thought that toxic constituents i) would be completely refractory to cloning,
or ii) exert their toxic effect upon induction, and so give poor protein yields.
Here, there seems to be an intermediate toxicity, such that I can consistently generate transformants,
but they just stop growing.

Has anyone else encountered this kind of problem?

I should mention that I tried growing the cells on LB + glucose (5%) plates.
I know that the pET system employs a lacUV5 promoter, which should not respond
to catabolite repression (although I'm afraid I don't understand why - is the CRP operator missing?)
but the Novagen people still suggest it can help in cases of toxicity.
Alas, cell growth was not augmented.

I look forward to any comments or suggestions.

Thanks,

David

dc.alexander at utoronto.ca









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