Deletion formation mutations in plasmid expression vectors are unfavored by runaway amplification conditions and differentially selected under kanamycin stress

Plasmid pCIneo is a ColE1-like mammalian expression vector also used as backbone for DNA vaccine development. We have recently shown that pCIneo spontaneously recombines due to the presence of two 28bp direct repeats. The persistence of low-frequency recombinants led us to evaluate the impact of environmental stresses typically found during plasmid production on plasmid copy number and recombination frequency. We observed an increase in pCIneo amplification (2.6-4.3-fold) in Escherichia coli cultures grown at 42 degrees C and also in minimal medium (at both 37 degrees C and 42 degrees C). These conditions fit to the smallest ratio between recombinant molecules and total plasmids. Conversely, increasing the dissolved oxygen tension from 20% to 40% in rich media did not have a significant impact on both plasmid copy number and recombination frequency, independently of the temperature used. We have also shown recently that the neomycin resistance (neo(r)) gene of pCIneo becomes actively transcribed as a result of recombination between the repeats. This prompted us to gain some insight into plasmid adaptation and competition by evaluating the impact of distinct concentrations of kanamycin on the differential selection of plasmid recombinant forms: monomer and heterodimers (1+2 and 1+3). We found the monomeric form to be predominantly recovered at lower concentrations of antibiotic whilst higher concentrations led to an increase in the percentage of the 1+2 form. The 1+3 heterodimeric form was invariably found at low percentages, independently of the concentration used.