Orientation of a PCR product in a T-vector

ChenHA Hzhen at freeuk.com
Fri Apr 5 13:34:27 EST 2002

Tatjana Schwabe wrote:
> That's what I always thought as well.
> Now, I actually already have this problem and I need the insert in the
> correct orientation (not in T-vector but later) - does anyone have an
> idea how to prevent E. coli from producing my protein and killing
> itself? It's under a cyanobacterial promotor which is apparently leaky,
> at least in DH5a. Is my only option now to try different strains?

If you want to express your protein in e.coli, one possibility would be
to clone it into something like pET.  Cloning wouldn't be a problem
because the protein would not be expressed in the absence of T7
polymerase.  When it comes to expression, you can either control the
expression very tightly (e.g. using pLysE cells), or grow cells without
T7 pol and induce the cells with phage containing the T7 pol.  There are
other vectors around which may be useful in such circumstances, hunt
around in literature or catalogues.  I haven't looked into this for some
time, and there must be many new vectors being produced that might be
useful for you.

> Thanks for ideas!
> Tatjana
> --
> Tatjana M.E. Schwabe
> Plant Biochemistry
> Ruhr-University Bochum

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