bacterial phage 3
TRANSCRIPT
BACTERIAL PHAGE 3
Replication/lifecycle of phage λ
Lytic replication
θ replication from the circular DNA molecule
which is bidirectional
Lysogenic replication
Integration into E. coli chromosome and
propagation of the “prophage”
Lytic “state” or “response” of bacteriophage λ
Lysogenic “state” or “response” of bacteriophage λ
Phage plaque assayExamples of properties of
different phage plaques
Genetic map of phage λ
Generation and purification of phage λ arms
Use of phage λ as a cloning vector
2 Types of vectors
1. Insertion:
1 site for inserting foreign DNA
2. Replacement:
2 sites for replacement of viral
sequences with inserted DNA
Assembly of phage λ particles
Screening a Lambda library
Screening a library of genomic DNA
The number of clones needed in order to have
all genomic sequences represented is calculated
as:
N = ln(1-P)/ln(1-f)
N = number of recombinants needed to have
a probability P of isolating a gene which
represents the fractional proportion of the
genome, f.
The average mammalian genome is 3x109 bp.
8.1x105 phage are therefore need to be screened
in order to have a 99% probability of isolating a
single copy gene in a 17kbp cloned segment.
(f = 1.7x104/3x109)
90mm dishes can accommodate up to 15,000
plaques.
150mm dishes can accommodate 50,000
plaques (17 plates).
Natural Log (ln)
M13 lifecycle
1. Bacteriophage P1
The largest of the common lysogenic phages
Lysogeny
a) Rarely integrated into the host genome
b) Exists as a plasmid with a copy number of approximately
one
c) Empirically has a linear genetic map because of frequent
site-specific recombination at the loxP locus, catalyzed by
the Cre protein
d) Inducible
3. Lysis
a) Forms concatamers by recombination, like T4
b) Packaged by the headful mechanism
c) Little degradation of host DNA
d) Many defective viruses produced: up to 20% of the
offspring have less than or equal to 40% of the genome
e) Defective viruses can reproduce at a high multiplicity
of infection
f) Very useful as a generalized transducing phage
2. Bacteriophage Mu
1. Most efficient transposon known
2. Produces mostly stable mutations
3. Bacteriophages P2 and P4: the odd couple
1. P2 is the larger of the two
a) Its genome is three times the size of that of P4
b) Its virion is slightly larger, also
There is no nucleotide sequence similarity between the
two genomes
However, their capsid proteins are identical! How can this
be?
P2 is a normal lysogenic phage
a) It has about 10 attachment sites in the host genome
b) Very difficult to induce, but does lyse host cells
naturally at low frequency
c) Rolling circle replication, similar to λ
P4 is not so normal…
a) Without P2, P4 will either stabilize as a high copy
number (30–50 copies) plasmid, or it will integrate, or it kills
the cell without successfully making progeny
b) With P2, P4 can perform a complete lytic cycle without
inducing P2.
(1) It commandeers all of P2’s capsid genes for its own
purposes.
(2) Two P4 genes are involved in the takeover.
c) P4 is a parasite of a parasite.