Sum7LS4Week4a(1slide)

Sum7LS4Week4a(1slide) - Bacteriophage Genetics: Mapping...

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Bacteriophage Genetics: Mapping Genes in Bacteriophage Chapter 8, pages 173 - 182, 185 - 188 Problems: 9, 12, 13, 16, 18, 19
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Phage-phage Recombination • Hershey experiment: -Two different phage genotypes can be recombined by simultaneously infecting host bacteria with two different types of phage and then screening progeny phage for recombinant genotypes. -Defines recombination frequency in phage. • Powerful experimental tool - one can measure very rare events
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• Spread E. coli cells on agar growth medium in a sterile petrie dish and grow at 37º C overnight. Phage Plaques -Eventually all of the bacteria in the vicinity of the original phage particle will be lysed yielding a clear spot (hole) in the lawn of bacteria, called a plaque. -Each overnight plaque contains ~1 x 10 8 bacteriophage. -Formation of “lawn” of bacteria on the surface of the medium. • If you add a single bacteriophage (T4 or T2), it will infect one bacterium, lyse it, and release 300 progeny phage about 25 minutes later…infecting neighboring bacteria (cycle repeats)
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• Most phage phenotypes are visualized as plaques on lawns of bacteria • Plaques can vary in morphology: - Large or small plaques are determined by how fast lysis occurs - Host range is a reflection of what strains of bacteria the phage can bind to and lyse Observing Phage
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• Wild type T4 phage produce small plaques with fuzzy margins, while rapid lysis (r) mutants produce large plaques with sharp margins. Rapid Lysis mutant (rapid lysis mutant) (wildtype) -When a T4 phage attaches to an E.coli cell that is already infected with wild-type T4 ( r + ), it delays lysis for up to 2 hours--a process called lysis inhibition fuzzy margins of wild-type plaques. -Lysis inhibition does not occur in bacteria infected with r mutants… all cells infected with r mutants all lyse rapidly (sharp-defined edges).
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• Usually bacteria infect a single strain of bacteria. -Some bacteriophage have acquired the ability to infect different host strains…these phages are known as host range mutants (h). Host range mutants • T2 wild-type and T2 h mutants can be distinguished by growing them on a mixed lawn of E.coli R and E.coli S cells. E.coli S T2 E.coli R T2 X T2 h T2 h
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Host range mutants Mutant (h) virus clear plaques because they infect and lyse all host cells--whether E.coli S or E.coli R. Wild-type (h + ) virus produce turbid plaques because they infect only S cells not the R cells…R cells are not lysed and can still grow in the region of the plaque.
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Cross: h - r + x h + r - E.coli S High multiplicity of infection (m.o.i.) : High concentration of phage. Can Phage Genomes Recombine? • Alfred Hershey and Max Delbrück, 1947: Traits: r + - small plaques r - - large plaques h + - infects E. coli S strain only h - - infects both E. coli S and R strain
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