fresh transformations for protein expression??

David Goodin dbg at scripps.edu
Sat Jan 27 18:09:50 EST 1996


Yes, I have also seen exactly this behavior for years in a
t7 expresion system for a nontoxic protein expressed at high
levels. You can even take an old culture and restreak to 
reselect resistant clones and it still wont express. However,
I can take that culture, isolate the plasmid, transform,
and expression is back. We see significant plasmid loss in
cultures even with heavy and continual amp selection, but
this may be something else due to the restreak phenom.
I don't know what it is... Only idea is that once cells
begin to express they grow slowly, but produce enough
lactamase to quickly degrade the amp in the media, then
quickly growing untransformed cells take over. Anyone have
a better undertanding?

Dave Goodin
The Scripps Research Institute

In article <DL0wEo.L78 at uns.bris.ac.uk>, bijgh at zeus.bris.ac.uk says...
Michael Szardenings (msz at bio.embnet.se) wrote:: 2. Many people are 
claiming, that these effects are due to plasmid
: instability. I have never seen any real proof for this, i.e. effects 
that
: go beyond the normal loss of plasmids due to selection for 
non-expressing
: clones. In the worst case I have seen growth arrest of the culture
: followed some time later by rapid growing cells without plasmid, as 
the
: ampicillin used in the experiment had been degraded during the initial
: growth.

I am expressing what I would expect to be non-toxic constructs using the 
pET system, and have had similar problems.  Freshly transformed cells 
express well but after a week siting in the fridge the cells won't 
express at all.  Even the glycerols I have made lost the ability to 
express protein.  Like Michael I'm not convinced that these problems are 
due to plasmid instability.  I've tried various experiments to test if 
the plasmid is unstable and have seen no evidence for it at all.  But if 
this loss of expression is not due to plasmid instability, what else 
could it be?

Any ideas?

Cheers,

Jared

--
Jared Head     at the Department of Biochemistry, University of Bristol




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