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Fundamental Biotechnology
Lecture# 6
Haji Akbar
M.Phil
Introduction Bacteriophage
Virus infecting Bacteria.
(eater of bacteria)
Short Phage
Groups on structure base
i. tailless
ii. Head with tail
iii. filamentous
Continue!!
Genome: (50% of total mass)
DNA (ssDNA or dsDNA)
or
RNA (ssRNA or ds RNA)
in tailless and tailed DNA is encapsulated in
capsid.
Historical:
Frederick Twort (1915) and Felix d'Herelle (1917)
were the first to recognize viruses which infect
bacteria, which d'Herelle called bacteriophages
(eaters of bacteria).
Diversity
There are at least 12 distinct groups of
bacteriophages, which are very diverse
structurally and genetically; the best known
ones are the common phages of E.coli
Virulent vs. Temperate Phages
Virulent phages: do not integrate their
genetic material into the host cell
chromosome and usually kill the host cells
(lytic infection) (e.g. T-phages of E.coli).
Temperate phages: may integrate into
the host DNA, causing LYSOGENY. (best
example λ phage)
Bacteriophage T4?
DNA is packaged in the head of T4 phage.
Genome of 173 kb of linear ds DNA.
During the early stages of a infection cycle T4
nucleases encoded by so called early genes
degrade the chromosomal DNA of E. coli in order to
obtain large quantities of nucleotide precursors for its
own DNA synthesis.
λ page
Genome 48.5 kb (46 genes)
Entire Genome has been sequenced and regulatory
site are known.
At the ends short (12bp) ss- complementary region
“cohesive or sticky” ends--- circulation after infection.
Region is known as cos site.
Phage infection begins!!
Adsorption
Lytic or lysogenic cycle depends on # of factors:
Nutrional & metabolic state of host cell
Multiplicity of infection (m.o.i- the ratio of page to
bacteria during adsorption)
In lysogenic phage genome integrates into host
chromroms =prophage
Packaged into mature phage
Bacteriophage infection can be easily monitored by plaque
formation. (in the lytic cycle, anyway)
Plaque forming Unit (p.f.u): is a measure of the number of particles
capable of forming plaques per unit volume (1,000 PFU/µl indicates that
there are 1,000 infectious virus particles in 1 µl of solution)
Page M13
Filamentous
ss-circular DNA (size 6407 bp)
DNA enter in to cell converted to double
stranded molecule known as replicative form
or RF.
Replicates until there are about 100 copies in
the cell.
Vectors based on Bacteriophage λ
The λ genome is 48.5 kb, in which 15 kb or so is
‘optional’
it contains genes that are only needed for
integration into the E. coli chromosome
(controlling lysogenic properties)
These segments can therefore be deleted
without impairing the ability of the phage to
infect bacteria and direct synthesis of new λ
particles by the lytic cycle.
Types of vector
Insertion vectors: (single recognition sit for
one or more restriction enzyme)
e.g. λgt10, Charon 16A. (both having EcoRI)
Replacement vectors:/ Substitution vector:
Having two restriction sits (RS) which flank a
region known as stuffer fragment (hatched)
e.g. EMBL4 and Chapron 40
λgt10: 7.5 kb (insert size), EcoRI lies in cI gene
which is basis of selection/ screening.
Charon 16A: 9kb, EcoRI lies in β-galactosidase gene
(lac Z).
EMBL4: 13.2 kb (stuffer fragment) and can clone 9 &
22 kb.
Chapron 40: (polystuffer) similar size fragment as
EMBL4
Vectors based on M13
Two aspect valuble for G.E.
1. RF similar to plasmid
2. ssDNA useful for sequencing by Dideoxy method.
507 bp intergenic region is the only part available for
manipulation.
Vectors constrict by introducing polylinker /lacZ αpeptide sequence into this region.
Mark reduction in cloning efficiency when DNA
fragment longer than 1.5 kb inserted.
Other vectors
Cosmid
Hybrid of plasmid/Phage vectors
contain E coli ori that allow it to maintained as a
plasmid in the cell and carries a λ cos site.
a plasmid that carries a λ cos site.
cos sites, act as substrates for in vitro packaging
because the cos site is the only sequence that a DNA
molecule needs in order to be recognized as a ‘λ
genome’ by the proteins that package DNA into λ
phage particles.
A cosmid can be 4-8kb in size, so up to 45-47 kb of
new DNA can be inserted.
inside the cell the cosmid cannot direct synthesis of
new phage particles and instead replicates as a
plasmid.
Recombinant DNA is therefore obtained from colonies
rather than plaques.
e.g:
pLFR-5 has two cos sites from page λ, separated by
ScaI RE sit, a multiple cloning site with six unique
restriction site origin of replication and Tetr gene
Cosmid Cloning System
 cos sites inserted
into a small
plasmid
Target DNA ligated
between two cosmid
DNA molecules
Recombinant DNA
packaged and E. coli
Infected as before
Can clone DNAs up
to 45 kb
Cosmid vectors
Advantages:



Useful for cloning very large DNA fragments
(32 - 47 kbp)
Inherent size selection for large inserts
Handle like plasmids
Disadvantages:

Not easy to handle very large plasmids (~ 50 kbp)
Phagemid vectors
plasmids which contain a small segment of
the genome of a filamentous phage, such as
M13, fd or f1.
contain all the cis-acting elements required
for DNA replication and assembly into phage
particles.
They permit successful cloning of inserts
several kilobases long.
Continue!!
Following transformation of a suitable E. coli strain
with a recombinant phagemid,
the bacterial cells are superinfected with a
filamentous helper phage, such as f1, which is
required to provide the coat protein.
Phage particles secreted from the superinfected cells
will be a mixture of helper phage and recombinant
phagemids.
The mixed single-stranded DNA population can be
used directly for DNA sequencing.
because the primer for initiating DNA strand
synthesis is designed to bind specifically to a
sequence of the phagemid vector adjacent to the
cloning site.
Commonly used phagemid vectors include the
pEMBL series of plasmids and the pBluescript family.
THE END