The document discusses several bacterial viruses (phages): 1. Phage λ can undergo lytic or lysogenic replication in E. coli and exists in either a lytic or lysogenic "state". 2. Phage P1 is the largest common lysogenic phage that can exist as a plasmid in its host genome. 3. Phage Mu is the most efficient known transposon. 4. Phages P2 and P4 have identical capsid proteins but different genomes. P2 is a normal lysogenic phage while P4 is a parasite that commandeers P2's capsid genes to complete its lytic cycle.

1. BACTERIAL PHAGE 3

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”

4. Lytic “state” or “response” of bacteriophage λ

5. Lysogenic “state” or “response” of bacteriophage λ

6. Phage plaque assay
Examples of properties of
different phage plaques

7. Genetic map of phage λ

8. Generation and purification of phage λ arms

9. 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

10. Assembly of phage λ particles

11. 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.

12. 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).

13. Natural Log (ln)

14. M13 lifecycle

17. 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

18. 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

19. 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

20. 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

21. 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.