A plasmid vector for isolation of strong promoters in Escherichia coli

In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for repl ication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. I nsertion in the MCS of DNA, fragments of Staphylococcus aureus bacteriophag es resulted in isolation of several clones very resistant to ampicillin. Th e DNA fragments inserted in these recombinant plasmids were sequenced and a ll of them contained putative promoter motifs. Direct measurement of the pe nicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. Accord ing to these results pProm is a powerful tool to perform studies on promote r strength and for industrial applications. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights r eserved.