Factors Affecting the Stability of E. coli Plasmid Vectors

Unlike natural plasmids, engineered plasmid vectors in general are not stably maintained in cell populations. This poses a great problem for industry where large scale fermentation of plasmid containing cells requires vast quantities of the appropriate antibiotics in order to select against plasmid-free segregants and therefore maximise product yield. Industrial applications apart, plasmid stability mechanisms constitute an interesting area of scientific research. Analysis of the stability properties of pAT153 and pBR322 multimers revealed that they were unstable in the AB1157 derivative strain DS903. Moreover, as the multimeric state of these plasmids increased, both copy number and stability progressively decreased. This implied that an origin-counting mechanism probably operates for ColE1-like plasmids and that partitioning at cell division is random. The probability of plasmid-free segregants arising from cells containing multimeric plasids was minimal on the basis of their calculated copy numbers. Therefore, several factors which could explain the instability of these plasmids were discussed, these include plasmid sequestration, copy number variance and competition between plasmid-free and plasmid-containing cells. Cloning the oar region of pSC101 into multimeric plasmids failed to increase their stability although it is likely that par can operate in the monomeric forms of these plasmids. From this, it was concluded that par cannot function in all plasmids and its location within the molecule may be important to its activity. Cloning the rom gene into the high copy number vector pUC8 reduced its copy number 6 fold but dramatically increased its stability. It is likely that cells possessing pUC8 (87 copies/genome equivalent) are at a competitive disadvantage to plasmid-free cells due to the metabolic burden imposed on them by pUC8. The stability of the rom+ pUC8 plasmid derivative (14 copies/genome equivalent) can be attributed to a decreased growth differential between p- and p+ cells, while the presence of the rom gene may lessen the probability of plasmid-free segregants arising by reducing copy number variance