Biomedical Engineering Reference
In-Depth Information
to isolate, M13 vectors (such as M13mp18) are used for DNA sequenc-
ing. However, because they are single stranded, matched pairs of the
M13 that produce either a + or - strand of the DNA (e.g., M13mp18 and
M13mp19) must be used in order to sequence DNA from both directions.
Sequencing from both directions is essential for accurate sequence de-
termination.
M13 phage do not contain a selectable antibiotic resistance marker.
However, M13 phage do contain a lecZ gene which contains the cloning
site. This makes it possible to distinguish bacteriophage that contain
DNA inserts from those that do not in the cloning process. The lacZ gene,
which is a component of the lac operon, encodes for the
enzyme. A substrate that can be used for is X-gal, which
turns blue when cleaved by As a result, phage in which the lacZ
gene has been disrupted by an insert will not make the enzyme, and
therefore any viral plaques produced will be clear rather than blue.
Screening methods using this technique are frequently referred to as
“blue-white” screening.
The lac operon holds a special place in the study of gene regulation
as it was the first genetic system that was understood in prokaryotic
cells, specifically E. coli. An operon is a set of contiguous genes that
are responsible for the synthesis of a particular gene product, often an
enzyme and is typical of prokaryotic cells. The operon contains both
structural genes and regulatory genes that function both to activate and
repress expression. The bacterium E. coli lives in lactose-rich environ-
ments such as the human gut, and like other cells requires glucose to
survive. E. coli can convert lactose to glucose using the enzyme
The lac operon consists of four genes, including lacZ, and can turn itself
on an off as required, a process known as genetic self-regulation. The
lac operon was discovered and its function elucidated in the 1960s by
Francois Jacob and Jacques Monod, who were awarded the Nobel Prize
in Medicine in 1965 for their important contributions to understanding
prokaryotic gene regulation.
Phage display
Bacteriophages can also be used to display antibody variable regions,
or other proteins or peptides, on their surface, a process known as phage
display. This is described in more detail in Chapter 4.
Specialized vectors
In addition to plasmids and phage vectors, there are other vectors that
have been developed for specialized use in DNA cloning. Commonly
used specialized vectors are described below.
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