Biology Reference
In-Depth Information
“Wild-type”
plasmids
are small DNA molecules that are stably inherited as extra-
chromosomal units in many kinds of bacteria. Plasmids are widely found in bacteria,
but usually are not essential to their host. Many plasmids carry genes for antibiotic
resistance, antibiotic production, heavy-metal resistance, an ability to degrade aro-
matic compounds, sugar fermentation, enterotoxin production, or hydrogen-sulfide
production. Most are covalently closed DNA circles, but some are linear.
Plasmids can be classified into two types depending upon whether they carry
a set of genes that promote
bacterial conjugation
. Plasmids also can be catego-
rized as to whether they are maintained in multiple copies in host cells or in
limited numbers per cell. Generally, plasmids that promote bacterial conjuga-
tion are relatively large and are present in one to three copies per bacterial cell.
Plasmids that do not promote bacterial conjugation are smaller and multiple
copies are found in a cell. Plasmids are “promiscuous” if they can promote their
own transfer to a wide range of bacteria and can be maintained stably in their
new hosts. Promiscuous plasmids can transfer cloned DNA molecules into differ-
ent bacteria. Wild-type plasmids could be used for cloning in
E. coli
, but they
suffer from several disadvantages, and genetically engineered plasmids have
been developed that have many desirable attributes.
The first genetic improvement of plasmids involved removing excess DNA
so that the plasmid is easier to manipulate
in vitro
, resistant to damage by
shearing, and readily isolated from bacterial cells. Smaller size is an advantage
because bacterial cells usually can sustain several smaller plasmids, which will
increase the yield of the recombinant DNA molecules.
A second improvement was the addition of one or more
selectable marker
genes to the plasmid (
Figure 5.2
). A selectable marker allows the experimenter
to identify those bacterial cells that have taken up the plasmid during the trans-
formation process. Many selectable markers are antibiotic resistance genes (for
example, ampicillin and tetracycline) that allow the transformed bacteria to be
grown on selective media.
A third improvement involved adding DNA containing a
multiple cloning site
or
polylinker
that can be cut by several restriction endonucleases (
Figure 5.3
).
The presence of these unique restriction or cloning sites is helpful because clon-
ing requires that both the vector and the exogenous DNA be cut with the same
endonuclease (or with endonucleases that produce the same kinds of ends) so
that the ends can be ligated together. If the plasmid had more than one site
that was cut by a specific endonuclease, the plasmid vector would be cut into
several fragments, resulting in defective vectors. A polylinker is a short segment
of DNA with sites where several different restriction endonucleases can cut.
