Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA

Abstract

DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage varphi80pt190 (trp(+)) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp(-) strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the varphi80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes.