A plasmid cloning vector with precisely regulatable copy number in Escherichia coli

We have developed a genetic system allowing for precise regulation of plasmid copy number in Escherichia coli cells. A cloning vector based on this system is described in this article. The pTC lambda3 plasmid is a lambda replicon, but transcription controlling initiation of plasmid DNA replication starts from the P-tetA promoter instead of phage lambda P-R promoter. Additionally, activity of P-tetA promoter is negatively controlled by the TetR repressor whose gene is located on the same plasmid vector and is induced by an analog of tetracycline, autoclaved chlortetracycline (aCT). Using different concentrations of the inducer it is possible to strictly regulate the copy number of pTC lambda3 and thus the copy number of a cloned gene. The usefulness of the system for the regulatable production of a protein encoded by a gene inserted into pTC lambda3 plasmid is demonstrated by dependence of beta -galactosidase activity on the lacZ gene dosage.