MICROBIAL GENETICS

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MICROBIAL GENETICS TRANSDUCTION
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MICROBIAL GENETICS - TRANSDUCTION
GENERALIZED TRANSDUCTION Two step procedure
Growth of phage on donor host producing some
transducing particles in the phage prep
Infection of recipient host by transducing
particles Phage P1, Growth of phage on donor
host Transducing Particles, Transducting phage
preps (lysates) Donor Host, Recipient
Host Infection of recipient culture by
transducing phage prep Recombination -
Homologous, Breaking and Rejoining Selection
Transductants, Recombinants SPECIALIZED
TRANSDUCTION Three step procedure Lysogenizati
on of donor host Induction of lysogenic culture
producing transducing particles Infection of
recipient host by transducing particles Phage
Lambda Infection of Donor Host Lysis or
Lysogeny Prophage Attachment Site, Prophage,
Lysogen Induction of lysogen, Lytic
growth Illegitimate recombination,
transducing particles, transducing phage
prep Infection of recipient culture,
Integration Selection Transductants Parti
al diploid transductants
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TRANSDUCTION PHAGES MOVE BACTERIAL GENES FROM
ONE HOST TO ANOTHER
GENERALIZED - ANY HOST GENE PHAGE GROWS ON
FIRST HOST (DONOR) SOME PHAGES PICK UP DONOR
CHROMOSOME FRAGMENT THESE PHAGES INFECT SECOND
HOST - THE RECIPIENT TRANSFER DONOR DNA INTO
RECIPIENT SPECIALIZED ONLY HOST GENES NEAR
PROPHAGE PHAGE LYSOGENIZES DONOR INDUCTION
SOME PHAGES PICK UP HOST (DONOR) DNA PHAGE
LYSOGENIZES SECOND HOST (RECIPIENT) CARRY DONOR
DNA
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TRANSDUCTION - GENERALIZED
A
GROW PI ON DONOR PHAGE P1 108 HOST E. COLI
108/ML WILD-TYPE LEUCINE DONOR
ADSORPTION PENETRATION SYNTHESIS
TAILS
P1 DNA
HEADS
LEU
ASSEMBLY
LEU
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LYSIS PRODUCES P1 PHAGE PREP
LEUCINE GENES OF HOST (DONOR)
PROGENY P1 PHAGES 1010/ ML
TRANSDUCING PARTICLE 107/ ML
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P1 RECIPIENT LYTIC GROWTH TRANSDUCING
PARTICLE RECIPIENT TRANSDUCTION
WILD-TYPE LEUCINE GENE
LEUCINE REQUIRING MUTANT RECIPIENT
MUTATION IN LEUCINE GENE
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HOMOLOGOUS RECOMBINATION NEAR LEFT AND RIGHT ENDS
FRAGMENT DEGRADED
WILD-TYPE LEUCINE TRANSDUCTANT- BINARY FISSION
PRODUCES ONLY LEU PROGENY
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DEMONSTRATION IN LAB
TRANSDUCING PHAGE PREP 107 P1 GROWN ON LEU
WILD-TYPE DONOR
MIXTURE 108 RECIPIENTS 107 P1 PHAGE 104
TRANSDUCING PARTICLES
108 LEU - RECIPIENT CELLS
PLATE
ON GLUCOSE (ONLY CARBON SOURCE) MEDIUM
LEU TRANSDUCTANTS
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SPECIALIZED TRANSDUCTION

• LYSOGEN FORMATION
• MIX l PREP
• AND GAL HOST
• WILD-TYPE

LYSIS - SOME CELLS LYSOGENY - SOME
CELLS ISOLATE LYSOGENS FOR NEXT STEP
GAL GALACTOSE LYSOGEN E. COLI (l )
- STRUCTURE
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GLUCOSE-6-PO4
GLYCOLYSIS
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PROPHAGE INDUCTION
DNA DAMAGE REPRESSOR CLEAVAGE LYTIC GENES
NO LONGER INHIBITED EXCISION, LYTIC GROWTH
PROGENY PHAGES, LYSIS
EXCISION
REPRESSOR FRAGMENT
EXCISIONASE
REPLICATED
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2. INDUCTION OF LYSOGEN l PREP
l AND lGAL
TRANSDUCING PARTICLES
l 1010 lGAL 106
GAL E. COLI (l) 108/ml
INDUCTION
INDUCTION - PROPHAGE LOOPS OUT
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ILLEGITIMATE RECOMBINATION
lGAL
HOST CHROMOSOME DELETED FOR GAL

PACKAGING lGAL INTO l HEAD CUTTING COS SITE TO
LINEARIZE
lGAL
HEAD PACKAGES
NOTE LYSIS OF INDUCED CULTURE l lGAL
lGAL
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3. MIX l PREP l lGAL GAL- E. COLI
RECIPIENT SELECT GAL TRANSDUCTANTS PLATE ON
GALACTOSE - SOLE CARBON SOURCE
GAL TRANSDUCTANTS
GALACTOSE MEDIUM ONLY CARBON
RECIPIENT ONLY 108 GAL-
PHAGE PREP ONLY 108 l 104 lGAL
MIX 108 l 104 lGAL 108 GAL- RECIPIENTS
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HOW l TRANSDUCING PARTICLE TRANSDUCES
LINEAR lGAL DNA IN PHAGE HEAD
CIRCULARIZES AFTER INJECTION
RECIPIENT GAL- CHROMOSOME
CUTTING BOP' AND BOB' FOR INTEGRATION
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INTEGRATION - JOINING THE CUT ENDS IN
NEW ARRANGEMENT
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lDNA
R A GAL
BOP'
BOB'
GENOTYPE? PHENOTYPE? DOMINANCE?