Biomedical Engineering Reference
In-Depth Information
Bacteriophage
l
Improved vectors derived from pBR322
Over the years, numerous different derivatives of
pBR322 have been constructed, many to fulfil
special-purpose cloning needs. A compilation of the
properties of some of these plasmids has been pro-
vided by Balbás
et al.
(1986).
Much of the early work on the improvement of
pBR322 centred on the insertion of additional unique
restriction sites and selectable markers, e.g. pBR325
encodes chloramphenicol resistance in addition
to ampicillin and tetracycline resistance and has a
unique
Eco
RI site in the
Cm
R
gene. Initially, each
new vector was constructed in a series of steps
analogous to those used in the generation of
pBR322 itself (Fig. 4.7). Then the construction of
improved vectors was simplified (Vieira & Messing
1982, 1987, Yanisch-Perron
et al.
1985) by the use
of
polylinkers
or
multiple cloning sites
(MCS), as
exemplified by the pUC vectors (Fig. 4.9). An MCS is
a short DNA sequence, 2.8 kb in the case of pUC19,
carrying sites for many different restriction endonu-
cleases. An MCS increases the number of potential
cloning strategies available by extending the range
of enzymes that can be used to generate a restriction
fragment suitable for cloning. By combining them
within an MCS, the sites are made contiguous, so
that any two sites within it can be cleaved simultan-
eously without excising vector sequences.
The pUC vectors also incorporate a DNA sequence
that permits rapid visual detection of an insert.
The MCS is inserted into the
lac
Z
Essential features
Bacteriophage λ is a genetically complex but very
extensively studied virus of
E. coli
(Box 4.1). Because
it has been the object of so much molecular-
genetical research, it was natural that, right from
the beginnings of gene manipulation, it should have
been investigated and developed as a vector. The
DNA of phage
, in the form in which it is isolated
from the phage particle, is a linear duplex molecule
of about 48.5 kbp. The entire DNA sequence has
been determined (Sanger
et al.
1982). At each
end are short single-stranded 5
λ
projections of 12
nucleotides, which are complementary in sequence
and by which the DNA adopts a circular structure
when it is injected into its host cell, i.e.
′
DNA
naturally has cohesive termini, which associate
to form the
cos
site.
Functionally related genes of phage
λ
are clus-
tered together on the map, except for the two posit-
ive regulatory genes
N
and
Q
. Genes on the left of the
conventional linear map (Fig. 4.10) code for head
and tail proteins of the phage particle. Genes of the
central region are concerned with recombination
(e.g.
red
) and the process of lysogenization, in which
the circularized chromosome is inserted into its host
chromosome and stably replicated along with it as
a prophage. Much of this central region, including
these genes, is not essential for phage growth and
can be deleted or replaced without seriously impair-
ing the infectious growth cycle. Its dispensability
is crucially important, as will become apparent
later, in the construction of vector derivatives of
the phage. To the right of the central region are
genes concerned with regulation and prophage
immunity to superinfection (
N
,
cro
,
c
I), followed by
DNA synthesis (
O
,
P
), late function regulation (
Q
)
and host cell lysis (
S
,
R
). Figure 4.11 illustrates the
λ
λ
′
sequence, which
encodes the promoter and the
-
galactosidase. The insertion of the MCS into the lacZ
α
-peptide of
β
′
fragment does not affect the ability of the
-peptide
to mediate complementation, but cloning DNA frag-
ments into the MCS does. Therefore, recombinants
can be detected by blue/white screening on growth
medium containing Xgal (see Box 3.2 on p. 35). The
usual site for insertion of the MCS is between
the iniator ATG codon and codon 7, a region that
encodes a functionally non-essential part of the
α
α
life cycle.
-complementation peptide. Recently, Slilaty and
Lebel (1998) have reported that blue/white colour
selection can be variable. They have found that
reliable inactivation of complementation occurs
only when the insert is made between codons 11
and 36.
Promoters and control circuits
As we shall see, it is possible to insert foreign DNA
into the chromosome of phage-
derivative and, in
some cases, foreign genes can be expressed effici-
ently via
λ
λ
promoters. We must therefore briefly
