bsteve@occrsh.ATT.COM (07/13/87)
We found that we could achieve copy numbers of near to 10,000 per cell by 16+ hour induction with cloramphenicol with ColE1 derived plasmids. The real problem in your application is that the expressivity of the plasmid genomes following the induction is very low due to the inhibitory effect of chloramphenicol on the tranlation system. Furthermore if you put the cells in rich media following the amplification induction, the amplification is lost. Your naturally occurring high copy number may be desirable if you wish do measure in-vivo tranlation products. Otherwise, the path to use for enzyme production is via in-vitro translation systems (which is a real pain in the neck). I would however wonder about the multiplicity of your enzyme's promoter and its cofactors, along with the cloned enzyme since these things are seldom co-linked and rather nasty to co-link. To make matters worse, the membrane binding sites for the amplified plasmids are often unamplified, leaving most of the plasmid genomes unexpressed. I think that your best bet is to use hot precursors for you enzyme assay. You may still not get higher activities due to the promoter problem. Steve Blasingame (AT&T Itasca, Ill.) ihnp4!gorgo!bsteve bsteve@gorgo.att.com