T7 promoters and expression

[Jim Woodgett]

In article <199406072215.PAA13126 at net.bio.net>, LKM100F at ODUVM.CC.ODU.EDU 
("Laura K. Moen") writes:

> []

> Has anyone else experienced such difficulties with T7 
> promoters?  I am surprised, but I am not exactly the most experienced 
> individual with protein expression.  Does anyone have any suggestions for 
> ways to suppress the background expression in our uninduced cells?  
> We have tried using JM109 and XL-1 Blue cells - similar problems in 
> either line.  

Have you tried pLysS (or pLysE) cells as originally described by Studier et 
al.?  These bacteria express lysozyme on a chloramphenicol selectable plasmid 
and have T7 polymerase intergated into the chromosome under an IPTG inducible 
promoter.  Lysozyme inhibits the basal levels of T7 polymerase thus cutting 
down the background.  These cells have the added advantage of being easily 
lysed by 3X freeze/thaw cycling which releases some lysozyme which then eats 
the cell wall.

I'm sure they're commercially available (but don't remember where from).

Jim