Sustainability and Stability - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 825

with a plasmid may leak sufficient levels of the auxotrophic factor that plasmid-free cells can
grow. With an antibiotic, the enzyme responsible for antibiotic degradation may leak into the
medium. Also, the enzyme may be so effective, even when retained intracellularly, that all the
antibiotics are destroyed quickly in a high-density culture, reducing the extracellular concen-
tration to zero. Although genes allowing the placement of selective pressure on a culture are
essential in vector development, the engineer should be aware of the limitations of selective
pressure in commercial-scale systems.
The other useful addition to plasmid construction is the addition of elements that
improve plasmid segregation. Examples are the so-called par and cer loci. These elements
act positively to ensure more even distribution of plasmids. The mechanisms behind
these elements are incompletely understood, although they may involve promoting
plasmid membrane complexes (the par locus) or decreasing the net level of multimeriza-
tion (the cer locus). Recall from Example 16.4 that multimerization decreases the number
of independent, inheritable units, thus increasing the probability of forming a plasmid-
free cell.
Any choice of vector construction must consider host cell characteristics. Proteolytic
degradation may not be critical if the host cell has been mutated to inactivate all known
proteases. Multimerization can be reduced by choosing a host with a defective recombination
system. However, host cells with a defective recombination system tend to grow poorly.
Many other possible host cell modifications enter into considerations of how to best construct
a vector for a commercial operation.
These qualitative ideas allow us to anticipate to some extent what problems may arise in
the maintenance of genetic stability and net protein expression. However, a good deal of
research has been done on predicting genetic instability.
16.5.6. HosteVector Interactions and Genetic Instability
Many of the structured mathematical models we discussed previously can be
extended to include component models for plasmid replication. Such models can then
predict how plasmid-encoded functions interact with the host cell. The quantitative
prediction of the growth-rate ratio and the development of plasmid-free cells due to seg-
regational losses can be readily made. The most sophisticated models will predict the
distribution of plasmids within a population and even the effects of multimerization
on genetic stability.
These models are too complex to warrant discussion in an introductory course. We will
consider some simple models that mimic many of the characteristics we discussed with
models of mixed cultures. A number of simple models for plasmid-bearing cells have been
proposed. The key parameters in such models are the relative growth rates of plasmid-free
and plasmid-containing cells and the rate of generation of plasmid-free cells (i.e. segrega-
tional loss). These parameters can be determined experimentally or even predicted for
more sophisticated models of host e vector interactions.
Let us consider how a simple model may be constructed and how the parameters of
interest may be determined experimentally.
The simplest model would be to consider only two cell types: plasmid free (X ) and
plasmid containing (X þ ). Furthermore, we assume that all plasmid-containing cells are