pET and ampicillin

pathology at mscf.med.upenn.edu pathology at mscf.med.upenn.edu
Wed Nov 23 18:25:58 EST 1994


In article <3anll5$hcl at mserv1.dl.ac.uk>, Clemens Suter-Crazzolara <un691cs at genius.embnet.dkfz-heidelberg.de> writes:
>> 	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


My suggestion is to make sure that the starter  liquid culture is never grown
to saturation; i.e., never use an overnight growing procedure for the starter.  
Instead, start with several colonies and grow for a few hours until the 2-3 
milliliter starter culture just begins to become hazy (of course, ampicillin is 
present).  Then add the starter to the full culture volume with 100microgram/ml
ampicillin.  The continuous presence of ampicillin should keep the selection 
pressure high enough to retain the plasmid through induction and protein
production.

Gregg Wells
Department of Pathology
University of Pennsylvania
Philadelphia, Pennsylvania  19104-4283
USA
email:  pathology at a1.mscf.upenn.edu




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