Stability of Recombinant Microorganisms

For segregational plasmid stability, cells need to be replicated so that the average number of plasmid copies per cell is doubled once per generation, and the plasmid copies need to be distributed equally to the daughter cells at cell division. It was reported that the problem of plasmid instability was encountered in systems employing E. coll (Kim and Ryu, 1984 Deretic et al., 1984 Caulcott et al., 1985), B. subtilis (Kadam et al., 1987), as well as yeast (Whiteway and Ahmed, 1984 Schwartz et al., 1988) as hosts. 

The fraction of cells carrying plasmid in the total population was shown to be a function of the number of generations of growth (Imanaka and Aiba, 1981). Therefore, as the size of the fermenter increases, the number of generations of growth increases, and the fraction of cell carrying plasmid and the product yield decrease. The problem of the plasmid instability is more serious if the large-scale fermenter is operated continuously. 

Estimate the number of generations of growth needed for genetically modified microorganisms from 1 mL culture to a 33,000L production-scale fermenter. Assume that the inoculum size in each stage of the 

For cells to grow from 3 percent to 100 percent of saturated cells, they have to multiply 100/3 = 33 times, which requires the cells to double n times as, 

Therefore, it takes about five generations. A typical inoculation steps with about 33 times volume increase can be as follows  

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