Sustainability and Stability - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 824

Multiple resistance genes are an aid in selecting for human-designed modifications of the
plasmid.
Another strategy for selection is to place on the vector the genes necessary to make an
essential metabolite (e.g. an amino acid). If the vector is placed in a host that is auxotrophic
for that amino acid, then the vector complements the host (as an auxotrophic mutant would
not be able to synthesize an essential compound on its own). In a mediumwithout that amino
acid, only plasmid-containing cells should be able to grow. Because the genes for the
synthesis of the auxotrophic factors can be integrated into the chromosome or because of
reversions on the parental chromosome, double auxotrophs are often used to reduce the
probability of nonplasmid-containing cells outgrowing the desired construction.
One weakness in both of these strategies is that, even when the cell loses the plasmid, the
plasmid-free cell will retain for several divisions enough gene product to provide antibiotic
resistance or the production of an auxotrophic factor (see Fig. 16.15 ). Thus, cells that will not
form viable colonies on selective plates (about 25 generations are required to form a colony)
can still be present and dividing in a large-scale system. These plasmid-free cells consume
resources without making product.
Another related problem, particularly in large-scale systems, is that plasmid-containing
cells may protect plasmid-free cells from the selective agent. For example, auxotrophic cells

death

c

death

c

c c

death

c

c

c c

c

death

c

death

c c

death

c

death

death

c

c

c

c

c c c

c

c

c

c c c

c

c

c

Normal
propagation

c c

c

c

c c

c c

c

FIGURE 16.15 Newly born plasmid-free cells usually contain sufficient complementing factor to with-
standing killing by a selective agent or starvation from the lack of a growth factor. In this case, the plasmid-free cell
undergoes three divisions before the complementing factor is reduced to an insufficient level leading to death.