Replication/lifecycle of phage λLytic replication θ replication from the circular DNA molecule which is bidirectionalLysogenic 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 assay Examples 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 vectors1. Insertion: 1 site for inserting foreign DNA2. Replacement: 2 sites for replacement of viral sequences with inserted DNA
Assembly of phage λ particles
Screening a Lambda libraryScreening a library of genomic DNAThe number of clones needed in order to haveall genomic sequences represented is calculatedas: 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 screenedin order to have a 99% probability of isolating asingle copy gene in a 17kbp cloned segment.(f = 1.7x104/3x109)90mm dishes can accommodate up to 15,000plaques.150mm dishes can accommodate 50,000plaques (17 plates).
Natural Log (ln)
1. Bacteriophage P1The largest of the common lysogenic phagesLysogeny 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 Mu1. Most efficient transposon known2. Produces mostly stable mutations3. Bacteriophages P2 and P4: the odd couple1. 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 thetwo genomesHowever, their capsid proteins are identical! How can thisbe?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 copynumber (30–50 copies) plasmid, or it will integrate, or it killsthe cell without successfully making progenyb) With P2, P4 can perform a complete lytic cycle withoutinducing 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.