Biomedical Engineering Reference
In-Depth Information
with 25 ml. Also, the use of antibiotics as selective agents may not be desirable in the large-
scale system, owing either to cost or to regulatory constraints on product quality.
In the next section, we will discuss some implications of these process constraints on
plasmid design. In the section following, we will discuss how simple mathematical models
of genetic instability can be constructed.
16.5.5. Considerations in Plasmid Design to Avoid Process Problems
When we design vectors for genetic engineering, we are concerned with elements that
control plasmid copy number, the level of target-gene expression, and the nature of the
gene product, and we must also allow for the application of selective pressure (e.g. antibiotic
resistance). The vector must also be designed to be compatible with the host cell.
Different origins of replication exist for various plasmids. The origin often contains tran-
scripts that regulate copy number. Different mutations in these regulatory transcripts will
yield greatly different copy numbers. In some cases, these transcripts have temperature
sensitive mutations, and temperature shifts can lead to runaway replication in which plasmid
copy number increases until cell death occurs.
Total protein production depends on both the number of gene copies (e.g. the number of
plasmids) and the strength of the promoter used to control transcription from these
promoters. Increasing copy number while maintaining a fixed promoter strength increases
protein production in a saturated manner. Typically, doubling copy numbers from 25 to 50
will double the protein production, but an increase from 50 to 100 will increase protein
production less than doubling. If the number of replicating units is above 50, pure segrega-
tional plasmid loss is fairly minimal. Most useful cloning vectors in Escherichia coli have
stable copy numbers from 25 to 250.
Many promoters exist. Some of the important ones for use in E. coli are listed in Table 16.3 .
An ideal promoter would be both very strong and tightly regulated. A zero basal level of
protein production is desirable, particularly if the target protein is toxic to the host cell. A
rapid response to induction is desirable, and the inducer should be cheap and safe. Although
temperature induction is often used on a small scale, thermal lags in a large fermentation
vessel can be problematic. Increased temperatures may also activate a heat-shock response
and increased levels of proteolytic enzymes. Many chemical inducers are expensive or might
cause health concerns if not removed from the product. Some promoters respond to starva-
tion for a nutrient (e.g. phosphate, oxygen, and energy), but the control of induction with
such promoters can be difficult to do precisely. The recent isolation of a promoter induced
by oxygen depletion may prove useful because oxygen levels can be controlled relatively
easily in fermenters.
Anytime a strong promoter is used, a strong transcriptional terminator should be used in
the construction. Recall from Chapter 10 that a terminator facilitates the release of RNA poly-
merase after a gene or operon is read. Without a strong terminator, the RNA polymerase may
not disengage. If the RNA polymerase reads through, it may transcribe undesirable genes or
may disturb the elements controlling plasmid copy numbers. In extreme cases, this might
cause runaway replication and cell death.
The nature of the protein and its localization are important considerations in achieving
a good process. To prevent proteolytic destruction of the target protein, a hybrid gene for
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