Post on 03-Jul-2015
description
Richa Banthia Ben Anderson
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
Overview
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
Formation of “sex pilus” or F-bridge between
cells
Attachment Via Pilli
Target DNA is replicated in F+
Genetic transfer of ssDNA to F-
Genetic Transfer of ssDNA
Complementary strands of DNA are
replicated in both F- and F+
Target DNA is incorporated into F- genome
Synthesis of
Complementary DNA
Cells separate
Form two F+ cells with completed recombinant plasmids
Both serve as donor cells for further conjugation
Separation and Formation
of Two F+ cells
Experiment 3
HFR (F+)
Able to produce
amino acids
o Thr, Leu, Thi
Sensitive to
Streptomycin
HFR strain used
for experimental
purposes
Mixed Culture
Requires amino
acids:
o Thr, Leu, Thi
Resistant to
Streptomycin
F-
Able to produce
Thr and Leu
o Thi not produced
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
F -
No Growth
Absence of
amino acids
Mixed Culture
Growth
Produced Thrand Leu
Antibiotic Resistance
F-HF
R
http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topic
s/plasmids/conjugation-mech.html
BIOL 3116 Advanced Microbiology Lab Manual