protein expression in PLysS

Kelvin Luther umluther at cc.umanitoba.ca
Sun Mar 28 13:58:21 EST 1999


Anant Patkar wrote:
> 
> We have also observed leaky expression in E. coli BL21(DE3) with
> pET23-based
> vector.  There was also significant plasmid stability problem.  Because
> of the leaky expression, only 60% of the cells would have plasmid after
> reaching stationary phase.  Has anyone seen this?
> 
> Anant Patkar
> University of Minnesota

I have seen my expression drop off  continually.  I have to keep
reclaiming my original transformants from -70 c to get the original
levels.  I tried the plasmid stability test in the pET manual and got no
useful information.  I have also tried retransforming with the original
plasmid sample, but have not been able to regain suitable expression
that way.  

Do you do retransformations, and pick clones from a plate for your
cultures or do you keep frozen cell stocks as a source of inoculum?


-- 
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
*Kelvin Luther 
*University of Manitoba                                             
*Department of Chemistry    "Illegitimati non carborundum"    
*Winnipeg, Manitoba           General Joseph W. Stilwell      
*Canada                                                     
* mailto:umluther at cc.umanitoba.ca                           
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~



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