The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli.
The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.
D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D009876:Operon; D010957:Plasmids; D012093:Replicon; D014364:Tryptophan / Q000235:genetics*
D004274:DNA, Recombinant; D014364:Tryptophan