This document discusses genetic variability and horizontal gene transfer in prokaryotes. It describes three main mechanisms of horizontal gene transfer - transduction, transformation, and conjugation. Conjugation involves the transfer of genetic material between bacteria through direct cell-to-cell contact via pilus. The process of conjugation is described, where the F+ donor cell transfers single-stranded DNA to the F- recipient cell. If the transfer is successful, it results in production of F+ recombinants that can serve as donor cells for further conjugation. An experiment demonstrating conjugation between HFR and F- E. coli strains on selective media is also summarized.

1.  
Richa Banthia Ben Anderson

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

6.  
Overview

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

12.  
Experiment 3

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

17. http://www.youtube.com/watch?v=O-EdX4MaMFE

18.  http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topic
s/plasmids/conjugation-mech.html
 BIOL 3116 Advanced Microbiology Lab Manual