TOOLS OF RECOMBINANT DNA

TECHNOLOGY
Department of Biosciences V.N.S.G.U. Surat
PHAGEMID
 A phagemid or phasmid is a DNA-based cloning vector, which has
both bacteriophage and plasmid properties. These vectors carry, in addition
to the origin of plasmid replication, an origin of replication derived from
bacteriophage.
 Phagemids are used in a variety of biotechnology applications; for example, they
can be used in a molecular biology technique called "Phage Display"

1) fusion proteins for a viral coat

protein + the gene to be evolved
(typically an antibody fragment)
are expressed in bacteriophage.
2) the library of phage are washed
over an immobilized target.
3) the remaining high-affinity
binders are used to infect bacteria.
4) the genes encoding the high-
affinity binders are isolated.
 5) those genes may have random mutations introduced and used to perform
another round of evolution. The selection and amplification steps can be performed
multiple times at greater stringency to isolate higher-affinity binders.

 A phagemid (plasmid + phage) is a plasmid that contains an f1 origin of

replication from an f1 phage. It can be used as a type of cloning vector in
combination with filamentous phage M13.
 A phagemid can be replicated as a plasmid, and also be packaged as single
stranded DNA in viral particles.
 Phagemids contain an origin of replication (ori) for double stranded replication, as
well as an f1 ori to enable single stranded replication and packaging into phage
particles.
 Many commonly used plasmids contain an f1 ori and are thus phagemids.
 Filamentous phages retard bacterial growth but, contrasting with the lambda
phage and the T7 phage, are not generally lytic. Helper phages are usually
engineered to package less efficiently (via a defective phage origin of
replication) than the phagemid so that the resultant phage particles contain
predominantly phagemid DNA.
 F1 Filamentous phage infection requires the presence of a pilus so only bacterial
hosts containing the F-plasmid or its derivatives can be used to generate phage
particles.
 Prior to the development of cycle sequencing, phagemids were used to generate
single stranded DNA template for sequencing purposes. Today phagemids are still
useful for generating templates for site-directed mutagenesis.
 Similarly to a plasmid, a phagemid can be used to clone DNA fragments and be
introduced into a bacterial host by a range of techniques, such
as transformation and electroporation.
 However, infection of a bacterial host containing a phagemid with a 'helper' phage,
for example VCSM13 or M13K07, provides the necessary viral components to
enable single stranded DNA replication and packaging of the phagemid DNA into
phage particles.
 The 'helper' phage infects the bacterial host by first attaching to the host cell's pilus
and then, after attachment, transporting the phage genome into the cytoplasm of
the host cell. Inside the cell, the phage genome triggers production of single
stranded phagemid DNA in the cytoplasm.
 This phagemid DNA is then packaged into phage particles. The phage particles
containing ssDNA are released from the bacterial host cell into the extracellular
environment.
 Detailed characterization of the filamentous phage life cycle and structural
features lead to the development of phage display technology, in which a range
of peptides and proteins can be expressed as fusions to phage coat proteins and
displayed on the viral surface.
 The displayed peptides and polypeptides are associated with the corresponding
coding DNA within the phage particle and so this technique lends itself to the
study of protein-protein interactions and other ligand/receptor combinations.
FF PHAGES
 Filamentous bacteriophage is a family of viruses (Inoviridae) that
infect bacteria. The phages are named for their filamentous shape, a worm-like
chain (long, thin and flexible, reminiscent of a length of cooked spaghetti),
about 6 nm in diameter and about 1000-2000 nm long.
 Ff phages (for F specific filamentous phages) is a group of almost
identical filamentous phage (genus Inovirus) including phages f1, fd, M13 and
ZJ/2, which infect bacteria bearing the F fertility factor
 The fertility factor (first named F by one of its discoverers Esther Lederberg;
also called the sex factor in E. coli or the F sex factor; also called F-
plasmid) allows genes to be transferred from one bacterium carrying the factor
to another bacterium lacking the factor by conjugation.
 The virion (virus particle) is a flexible filament measuring about 6 by 900 nm,
comprising a cylindrical protein tube protecting a single-stranded circular DNA
molecule at its core.
 The phage codes for only 11 gene products, and is one of the simplest viruses
known. It has been widely used to study fundamental aspects of molecular
biology.
 George Smith and Greg Winter used f1 and fd for their work on phage
display for which they were awarded a share of the 2018 Nobel Prize in
Chemistry.
M13
 M13 is one of the Ff phages (fd and f1 are others), a member of the
family filamentous bacteriophage (inovirus). Ff phages are composed of
circular single-stranded DNA (ssDNA) which is 6407 nucleotides long
encapsidated in approximately 2700 copies of the major coat protein p8, and
capped with about 5 copies each of four different minor coat proteins (p3 and
p6 at one end and p7 and p9 at the other end).
 The minor coat protein p3 attaches to the receptor at the tip of the F pilus of the
host Escherichia coli. The life cycle is relatively short, with the early phage
progeny exiting the cell ten minutes after infection. Ff phages are chronic
phage, releasing their progeny without killing the host cells.
 The infection causes turbid plaques in E. coli lawns, of intermediary opacity in
comparison to regular lysis plaques. However, a decrease in the rate of cell
growth is seen in the infected cells.
 M13 plasmids are used for many recombinant DNA processes, and the virus
has also been used for phage display, directed evolution, nanostructures and
and nanotechnology applications.

Blue: Coat Protein pIII; Brown: Coat

Protein pVI; Red: Coat Protein pVII;
Limegreen: Coat Protein pVIII; Fuchsia:
Coat Protein pIX; Purple: Single
Stranded DNA