Construction of a Co1E1 plasmid bearing inducible high-copy-number phenotype.

In order to construct plasmids bearing inducible high-copy-number phenotype, the cloning plasmid pBR322 was modified as follows: a DNA fragment containing a strong synthetic promoter (P1), synthetic lac operator (O1), DNA sequence corresponding to the RNAI/RNAII region of the Co1E1 replicon and the CAT gene transcription terminator was substituted for the 29 bp EcoRI/HindIII DNA fragment. Two types of plasmids were constructed in this way, differing in the orientation of the RNAI/RNAII fragment. Depending on the orientation these plasmids coded for RNA molecules representing either RNAI or RNAII domains. It was found that when RNAII molecules were overproduced the plasmid copy number was about 4 times higher than that of pBR322 and only negligible change in the plasmid copy-number value was observed upon overproduction of RNAI molecules.