Biomedical Engineering Reference
In-Depth Information
CHAPTER 4
Basic biology of plasmid
and phage vectors
The enzymatic interconversion of supercoiled, relaxed
CCC DNA* and OC DNA is shown in Fig. 4.1.
Because of their different structural configurations,
supercoiled and OC DNA separate upon electro-
phoresis in agarose gels (Fig. 4.2). Addition of an
intercalating agent, such as ethidium bromide, to
supercoiled DNA causes the plasmid to unwind. If
excess ethidium bromide is added, the plasmid will
rewind in the opposite direction (Fig. 4.3). Use of
this fact is made in the isolation of plasmid DNA
(see p. 48).
Not all plasmids exist as circular molecules.
Linear plasmids have been found in a variety of bac-
teria, e.g.
Streptomyces
sp. and
Borrelia burgdorferi
.
To prevent nuclease digestion, the ends of linear
plasmids need to be protected and two general
mechanisms have evolved. Either there are repeated
sequences ending in a terminal DNA hairpin loop
(
Borrelia
) or the ends are protected by covalent
attachment of a protein (
Streptomyces
). For more
details of linear plasmids the reader should consult
Hinnebusch and Tilly (1993).
Plasmids are widely distributed throughout the
prokaryotes, vary in size from less than 1
Plasmid biology and
simple plasmid vectors
Plasmids are widely used as cloning vehicles but,
before discussing their use in this context, it is appro-
priate to review some of their basic properties.
Plasmids are replicons which are stably inherited in
an extrachromosomal state. Most plasmids exist as
double-stranded circular DNA molecules. If both
strands of DNA are intact circles the molecules are
described as covalently closed circles or CCC DNA
(Fig. 4.1). If only one strand is intact, then the
molecules are described as open circles or OC DNA.
When isolated from cells, covalently closed circles
often have a deficiency of turns in the double helix,
such that they have a supercoiled configuration.
Supercoiled
DNA
DNA gyrase
Endonuclease
10
6
Topo-
isomerase
×
10
6
, and are generally
dispensable. Some of the phenotypes which these
plasmids confer on their host cells are listed in
Table 4.1. Plasmids to which phenotypic traits have
not yet been ascribed are called
cryptic
plasmids.
Plasmids can be categorized into one of two major
type - conjugative or non-conjugative - depending
upon whether or not they carry a set of transfer
genes, called the
tra
genes, which promote bacterial
conjugation. Plasmids can also be categorized on the
basis of their being maintained as multiple copies per
daltons to greater than 200
×
Endonuclease
DNA ligase
Relaxed, covalently
closed circular DNA
Open, circular DNA
* The reader should not be confused by the terms
relaxed circle
and
relaxed plasmid.
Relaxed circles are CCC DNA that does not
have a supercoiled configuration. Relaxed plasmids are plas-
mids with multiple copies per cell.
Fig. 4.1
The interconversion of supercoiled, relaxed
covalently closed circular DNA and open circular DNA.
