Biomedical Engineering Reference
In-Depth Information
essential in genetic engineering). A cell marks its own DNA (e.g. through methylation of
certain purine or pyrimidine bases) to distinguish it from foreign DNA. These modifications
block the action of a cell's own restriction endonucleases on its own DNA. Under natural
conditions, gene transfer is effective only if the donor DNA is from the same or closely related
species.
The entrance of donor DNA into a cell holds the key for genetic recombination. The three
mechanisms of foreign DNA entering a cell are discussed in the following three sections.
14.3.2. Transformation
The uptake of naked DNA cannot be done by all genera of bacteria. Even within trans-
formable genera, only certain strains are transformable (
competent
). Competent cells have
a much higher capacity for binding DNA to the cell surface than do noncompetent cells.
Competency can depend on the physiological state of the cell (current and previous growth
conditions). Even in a competent population, only a small fraction of cells are transformable.
Typically, about 0.1
e
1.0% is transformable.
Noncompetent cells may be rendered competent under special conditions (a genetic
engineering tool).
Escherichia coli
are not normally competent, but their importance to
microbial genetics has led to the development of empirical procedures to induce compe-
tency. This procedure involves treating
E. coli
with a high concentration of calcium ions
coupled with temperature manipulations. The competency of treated cells varies among
strains of
E. coli
, but is typically rather low (about one in a million cells becomes success-
fully transformed). With the use of selective markers, this frequency is still high enough
to be quite useful.
Transformation is useful only when the information that enters the cell can be propagated.
When doing transformation, we typically use a vector called a
plasmid
. This element forms
the basis for most industrially important fermentations with recombinant DNA. A plasmid
is an autonomous, self-replicating, double-strand piece of DNA that is normally extrachro-
mosomal. Some plasmids are maintained as low copy number (20
e
100 copies per cell). These
plasmids differ in their mechanisms for partitioning at cell division and in the control of their
replication. Plasmids encode genes typically for proteins that are nonessential for growth, but
that can confer important advantages to their host cells under some environmental circum-
stances. For example, most plasmids encode proteins that confer resistance to specific antibi-
otics. Such antibiotic resistance is very helpful in selecting for cells that contain a desired
plasmid.
14.3.3. Transduction
DNA transfer from one cell to another can be mediated by viruses and certainly plays an
important role in nature. In the most common type of
transduction
, non-integration transduc-
tion, infection of a recipient cell results in fragmentation of the bacterial DNA into 100 or so
pieces. One of these fragments can be packaged accidentally into a phage particle during
formation (the bottom process in
Fig. 14.4
). The defective phage particle then injects bacterial
DNA into another cell, where it can recombine with that cell's DNA. With non-integration
transduction, any bacterial gene may be transferred.
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