 

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
 Antibioticresistance
 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 The Recipient The Conjugate
 F+  F-  HFR
o Fertility factor o No fertility o High
 Contains
factor frequency
 Receives
recombinant
genetic
material for genetic  Resulting

gene material genetic

exchange  “Female” cell
recombinant
 “male” cell  Transfers
entire genomic
DNA when
 Attachment Via Pilli

 Formation of “sex pilus” or F-bridge between

cells
 Genetic Transfer of ssDNA

 TargetDNA 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

 Cells separate
 Form two F+ cells with completed recombinant plasmids
 Both serve as donor cells for further conjugation
  
Experiment 3
 HFR (F+) F- Mixed Culture
 Able to produce  Requires amino  Able to produce
amino acids acids: Thr and Leu
o Thr, Leu, Thi o Thr, Leu, Thi o Thi not produced

 Sensitive to  Resistant to  Resistant to

Streptomycin Streptomycin Strepromycin
 HFR strain used
for experimental
purposes
 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+) F- Mixed Culture
 No growth  No Growth  Growth

 Sensitivity to  Absence of  Produced Thr

Antibiotics amino acids and Leu

 Antibiotic
Resistance

HF F-
R
http://www.youtube.com/watch?v=O-EdX4MaMFE
 http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topic
s/plasmids/conjugation-mech.html
 BIOL 3116 Advanced Microbiology Lab Manual