Bacterial phage 3

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BACTERIAL PHAGE 3

Transcript of Bacterial phage 3

Page 1: Bacterial phage 3

BACTERIAL PHAGE 3

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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”

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Lytic “state” or “response” of bacteriophage λ

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Lysogenic “state” or “response” of bacteriophage λ

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Phage plaque assayExamples of properties of

different phage plaques

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Genetic map of phage λ

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Generation and purification of phage λ arms

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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

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Assembly of phage λ particles

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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.

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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).

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Natural Log (ln)

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M13 lifecycle

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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

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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

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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

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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

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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.