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MICROBIAL GENETICS TRANSDUCTION

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 Lysogenization 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 Partial diploid transductants

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

TRANSDUCTION - GENERALIZED
A

GROW PI ON DONOR PHAGE P1 ~108 HOST E. COLI ~108/ML WILD-TYPE LEUCINE+ [DONOR]
TAILS HEADS
LEU+

ADSORPTION PENETRATION SYNTHESIS

P1 DNA

ASSEMBLY

LEU+

LYSIS PRODUCES P1 PHAGE PREP LEUCINE GENES OF HOST (DONOR) PROGENY P1 PHAGES ~1010/ ML TRANSDUCING PARTICLE ~107/ ML

6 INFECT RECIPIENT WITH P1 (GROWN ON WILD-TYPE LEU + DONOR) RECIPIENT IS LEUCINE-REQUIRING MUTANT P1 PHAGE PREP CONTAINS P1 AND TRANSDUCING PARTICLES

P1 + RECIPIENT LYTIC GROWTH TRANSDUCING PARTICLE + RECIPIENT TRANSDUCTION

WILD-TYPE LEUCINE GENE

LEUCINE REQUIRING MUTANT [RECIPIENT]

= MUTATION IN LEUCINE GENE

HOMOLOGOUS RECOMBINATION NEAR LEFT AND RIGHT ENDS

WILD-TYPE LEUCINE+ TRANSDUCTANTBINARY FISSION PRODUCES ONLY LEU+ PROGENY

FRAGMENT DEGRADED

DEMONSTRATION IN LAB

PLATE

~108 LEU RECIPIENT CELLS

TRANSDUCING PHAGE PREP ~107 P1 [GROWN ON LEU + WILDTYPE DONOR]

MIXTURE: ~108 RECIPIENTS ~107 P1 PHAGE ~104 TRANSDUCING PARTICLES

ON GLUCOSE (ONLY CARBON SOURCE) MEDIUM

LEU + TRANSDUCTANTS

SPECIALIZED TRANSDUCTION
1. LYSOGEN FORMATION MIX l PREP AND GAL+ HOST [WILD-TYPE] GAL = GALACTOSE LYSOGEN = E. COLI (l +) STRUCTURE LYSIS - SOME CELLS LYSOGENY - SOME CELLS [ISOLATE LYSOGENS FOR NEXT STEP]

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GLUCOSE-6-PO4
GLYCOLYSIS

GALACTOSE + ATP

GLUCOSE-1-PO4 + ADP

GALACTOSE OPERON
TRANSCRIPTION START

REPRESSOR

STRUCTURAL GENES3 ENZYMES

GAL+ PHENOTYPE - CAN USE GALACTOSE AS SOLE CARBON SOURCE GAL- MUTANT - WILL NOT GROW ON GALACTOSE AS SOLE CARBON SOURCE [GROWS ON GLUCOSE]

PROPHAGE INDUCTION
DNA DAMAGE REPRESSOR CLEAVAGE LYTIC GENES NO LONGER INHIBITED EXCISION, LYTIC GROWTH PROGENY PHAGES, LYSIS EXCISION

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REPRESSOR FRAGMENT

EXCISIONASE

REPLICATED

2. INDUCTION OF LYSOGEN l+ AND lGAL+

l PREP

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TRANSDUCING PARTICLES

GAL+ E. COLI (l+) ~108/ml

INDUCTION

l+ ~1010 lGAL+ ~106

INDUCTION PROPHAGE LOOPS OUT

13 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+

3. MIX: l PREP [l+; lGAL+] GAL- E. COLI RECIPIENT SELECT GAL+ TRANSDUCTANTS PLATE ON GALACTOSE - SOLE CARBON SOURCE
GAL+ TRANSDUCTANTS

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GALACTOSE MEDIUM [ONLY CARBON]

RECIPIENT ONLY ~108 GAL-

PHAGE PREP ONLY ~108 l+; ~104 lGAL+

MIX: ~108 l ~104 lGAL+ ~108 GAL-

RECIPIENTS

HOW l TRANSDUCING PARTICLE TRANSDUCES

LINEAR lGAL+ DNA IN PHAGE HEAD CIRCULARIZES AFTER INJECTION

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RECIPIENT GALCHROMOSOME
CUTTING BOP' AND BOB' FOR INTEGRATION

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INTEGRATION - JOINING THE CUT ENDS [IN NEW ARRANGEMENT]

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lDNA

R BOP'

GAL+ BOB'

GENOTYPE? PHENOTYPE? DOMINANCE?