Download Bacterial Conjugation

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 Genetic
variability – ESSENTIAL!!!
o Survival of the species
 Diploid
Eukaryotes
o Crossing over
o Meiosis
 Haploid
Prokaryotes
o Transduction
o Transformation
o Conjugation
 Genetic
material exchanged via Viruses
o E.g. bacteriophage lambda
 No
physical contact b/w bacteria
 Promotes drug resistance
 Horizontal gene transfer
 Acquisition
of exogenous DNA fragments from
environment
o E.g. Plasmids
 Antibiotic
resistance
 Pesticide resistance in crops
o E.g. Agrobacterium tumefaciens
 Horizontal
gene transfer
 Exchange
of genetic material b/w bacteria
 Physical contact required
 DNA exchanged via Pilli
 Drug resistance!!
 Horizontal Gene Transfer
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Overview
The Donor
 F+
o Fertility factor
 Contains
genetic
material for
gene exchange
 “male” cell
The Recipient
 F-
The Conjugate
 HFR
o No fertility
factor
o High frequency
recombinant
 Receives
 Resulting
genetic
material
 “Female” cell
genetic
recombinant
 Transfers entire
genomic DNA
when mixed
with F- cells
Attachment Via Pilli
 Formation
of “sex pilus” or F-bridge between cells
Genetic Transfer of ssDNA
 Target
DNA is replicated in F+
 Genetic transfer of ssDNA to F-
Synthesis of Complementary
DNA
 Complementary
strands of DNA are
replicated in both F- and F+
 Target DNA is incorporated into F- genome
Separation and Formation of
Two F+ cells
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Cells separate
Form two F+ cells with completed recombinant plasmids
Both serve as donor cells for further conjugation
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Experiment 3
HFR (F+)
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Able to produce
amino acids

o Thr, Leu, Thi
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Sensitive to
Streptomycin
HFR strain used
for experimental
purposes
Mixed Culture
FRequires amino
acids:

o Thi not produced
o Thr, Leu, Thi

Resistant to
Streptomycin
Able to produce
Thr and Leu

Resistant to
Strepromycin
M9ST Agar
 Minimal Media 9
 Streptomycin
o Antibiotic formation
o Inhibition of F- growth
 Thiamine
o Inhibition of F+ growth
o Promotion of HFR growth
 Mix
F- and HFR (F+) strains
 Incubation in 37o water bath for proper conjugation
o 30 minutes*
 Vortex
to terminate genetic transfer
 Centrifugation – DNA Pellet formation
o Removal of supernatant (TSB broth)*
 Resuspension
with M9 Broth
 Plated on M9ST Agar plates
 Incubation at 37o for 48hrs
HFR (F+)
 No growth
 Sensitivity to
Antibiotics
HFR
F No Growth
 Absence of
amino acids
F-
Mixed Culture
 Growth
 Produced Thr
and Leu
 Antibiotic
Resistance
http://www.youtube.com/watch?v=O-EdX4MaMFE
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http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topic
s/plasmids/conjugation-mech.html
BIOL 3116 Advanced Microbiology Lab Manual