Bacterially Expressed Proteins: IPTG induction

Ted Michelini tedm at darkwing.uoregon.edu
Mon Aug 26 20:11:17 EST 1996


In article <321C9554.6A91 at lac.jci.tju.edu>, "Jay L. Rothstein"
<rothstei at lac.jci.tju.edu> wrote:

> We have cloned several pieces of genes into the invitrogen pTRCHis
> vector and have expressed them in DH10b, BL21 and similar bacteria.  The
> problem we have is that for some unknown reason we are unable to
> consistently induce these genes with IPTG.  Some days they induce fine
> other they do not (only background proteins can be seen)  We cannot find
> a correlation with  any feature of bacterial induction.  Does anyone
> have experience with this intermittant start/stop expression in a
> particular clone.  This can even occur in situations where one induction
> works but the next day it doesn't with the same bacteria (taken froma
> frozen stock or from a plate).  We have also altered IPTG stocks and
> levels but it does not appear to be cause by the IPTG.  Also the same
> vector in DH10B or XL1 blue or BL21 cells behave the same way in that we
> will intermittantly see no IPTG induction. While we CAN get induction
> this inconsistency is annoying and wasteful.  Any suggestions?  Has
> anyone experineced this probelm with bacterial expression vectors?
> -- 
> __________________________________________________________
> 
> Jay L. Rothstein, Ph.D.   

Jay,  I have had similar inconsistancies lately and I think Zheng may be
right, plasmid instability could be the reason. Remember that true
repression of the Tac promoter seldom occurs and the plasmid may be
strongly selected against during growth with a leaky promoter. Perhaps
promoter mutations/attenuations are selected for(?). Adding glucose to the
growth media and using more stable selective agent (Carb/Kan) should help.
Its also very easy to take a couple of serial dilutions at inductions and
harvest and plate on selective and non-selective media to see plasmid
retention.Also lac Iq strains need more IPTG but you're adding a ton
anyway... Good Luck

Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu



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