2. Genetic variability – ESSENTIAL!!!
o Survival of the species
Diploid Eukaryotes
o Crossing over
o Meiosis
Haploid Prokaryotes
o Transduction
o Transformation
o Conjugation
3. Genetic material exchanged via Viruses
o E.g. bacteriophage lambda
No physical contact b/w bacteria
Promotes drug resistance
Horizontal gene transfer
4. 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
5. Exchange of genetic material b/w bacteria
Physical contact required
DNA exchanged via Pilli
Drug resistance!!
Horizontal Gene Transfer
7. The Donor
F+
o Fertility factor
Contains
genetic
material for
gene
exchange
“male” cell
The Recipient
F-o
No fertility
factor
Receives
genetic
material
“Female” cell
The Conjugate
HFR
o High
frequency
recombinant
Resulting
genetic
recombinant
Transfers
entire genomic
DNA when
mixed with F-cells
8. Formation of “sex pilus” or F-bridge between
cells
Attachment Via Pilli
9. Target DNA is replicated in F+
Genetic transfer of ssDNA to F-Genetic
Transfer of ssDNA
10. Synthesis of
Complementary DNA
Complementary strands of DNA are
replicated in both F- and F+
Target DNA is incorporated into F- genome
11. Cells separate
Separation and Formation
of Two F+ cells
Form two F+ cells with completed recombinant plasmids
Both serve as donor cells for further conjugation
13. HFR (F+)
Able to produce
amino acids
o Thr, Leu, Thi
Sensitive to
Streptomycin
HFR strain used
for experimental
purposes
Mixed Culture
F-
Requires amino
acids:
o Thr, Leu, Thi
Resistant to
Streptomycin
Able to produce
Thr and Leu
o Thi not produced
Resistant to
Strepromycin
14. 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
15. 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
16. HFR (F+)
No growth
Sensitivity to
Antibiotics
F -
No Growth
Absence of
amino acids
Mixed Culture
Growth
Produced Thr
and Leu
Antibiotic
Resistance
HF F-R
