Enzyme and Microbial Technology, Vol.16, No.3, 240-246, 1994
Stability of a Recombinant Shuttle Plasmid in Bacillus-Subtilis and Escherichia-Coli
The recombinant shuttle plasmid pCPPS-31, which confers carboxymethylcellulase (CMCase) production and neomycin resistance (Ne-r), was segregationally but not structurally unstable in both B. subtilis (50-60%) and Escherichia coli (80-90%) when grown in serial batch cultures for 72 h diluting 1:1,000 with fresh medium every 24 h. The plasmid showed 80-100% segregational and complete structural stability in B. subtilis during batch, fed-batch, and continuous culture e fermentations in minimal M9 medium. In E. coli the instability was greater than or equal to 50% in 36 h of batch or continuous culture and approached 100% in 72 h of continuous fermentation. The plasmid was more stable (greater than or equal to 60%) during fed-batch fermentation. Stability in B. subtilis and instability in E. coli map be attributed to (i) the significant growth-rate advantage of plasmid-free (P-) cells over plasmid-harboring (P+) cells in the case of E. coli, an advantage that was absent in B. subtilis, and (ii) the existence of plasmid in oligomeric forms in E. coli and in mainly monomeric forms in B. subtilis.
Keywords:SEGREGATIONAL INSTABILITY;CONTINUOUS CULTURE;COPY NUMBER;CLONING;DNA;STABILIZATION;CONSTRUCTION;EXPRESSION;BACTERIA;GENES