pET and ampicillin

Todd Miller - Pharmacology tmiller at newssun.med.miami.edu
Thu Nov 10 09:11:56 EST 1994


	Our lab is using the pET system to express eukaryotic pro-
teins.  We seem to be experiencing a problem with the BL21 host cells
losing their plasmid.  Does anyone have suggestions on how to avoid
this?  We're thinking of trying carbenicillin but its about 10 times
as expensive as ampicillin.  Also, have inhibitors of beta lactamase
ever been used (like clavulanic acid)?

	I'm aware that overnight cultures should be highly diluted and
we've also played around a bit with the pLys S and E containing hosts.
We're trying to use this system in a fermentor, but I fear that if we
grow to the high densities that can be achieved in a fermentor, none
of the bacteria will have the plasmid when we induce expression.

Thanks for any suggestions.


Todd Miller
tmiller at newssun.med.miami.edu




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