pET and ampicillin

wch190 at lulu.acns.nwu.edu wch190 at lulu.acns.nwu.edu
Sat Nov 26 15:10:15 EST 1994


In article <1994Nov23.182558.1 at mscf.med.upenn.edu>,
pathology at mscf.med.upenn.edu wrote:

> 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

Another thing you can do is pellet down the cells of the overnight culture
wash twice with LB to wash off a ll the beta lactamase and then use as a
starter culture.

Venita de Almeida 
venita at merle.acns.nwu.edu



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