pET and ampicillin

[Richard James]

> 	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)?
> 
You do not say if the plasmid loss happens with your pET vector
 control; ie. without inserts. Assuming that it does not then your
 inserted gene(s) are lethal to the host cell. You must use pLysS etc
 to suppress their transcription.


Clavulanic acid would be no good as it would prompt killing of
 plasmid-containing cells by ampicillin. Carbenicillin is no better
 than ampicillin in countering the problem of lethality of the
 inserted gene.

Good luck.

Richard James


>