Bacterial Conjugation

Bacterial Conjugation

1. Bacterial Conjugation Richa Banthia Ben Anderson

2. Genetic recombination Genetic variability – ESSENTIAL!!! Survival of the species Diploid Eukaryotes Crossing over Meiosis Haploid Prokaryotes Transduction T ransformation Conjugation

3. Transduction Genetic material exchanged via Viruses E.g. bacteriophage lambda No physical contact b/w bacteria Promotes drug resistance Horizontal gene transfer

4. Transformation Acquisition of exogenous DNA fragments from environment E.g. Plasmids Antibiotic resistance Pesticide resistance in crops E.g. Agrobacterium tumefaciens Horizontal gene transfer

5. Conjugation Exchange of genetic material b/w bacteria Physical contact required DNA exchanged via Pilli Drug resistance!! Horizontal Gene Transfer

6. Bacterial Conjugation Overview

7. Terminology The Donor F+ Fertility factor Contains genetic material for gene exchange “male” cell The Recipient F- No fertility factor Receives genetic material “Female” cell The Conjugate HFR High frequency recombinant Resulting genetic recombinant Transfers entire genomic DNA when mixed with F- cells

8. 1. Cell – Cell C ontact Formation of “sex pilus ” or F-bridge between cells Attachment Via P illi

9. 2. Mobilization Target DNA is replicated in F+ G enetic transfer of ssDNA to F- Genetic Transfer of ssDNA

10. 3. DNA Synthesis Complementary strands of DNA are replicated in both F- and F+ Target DNA is incorporated into F- genome Synthesis of Complementary DNA

11. 4. Separation 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

12. Bacterial Conjugation in E. coli Experiment 3

13. E. c oli Strains HFR (F+) Able to produce amino acids T hr , L eu , T hi Sensitive to S treptomycin HFR strain used for experimental purposes Mixed Culture Requires amino acids: T hr , Leu , Thi Resistant to S treptomycin F- Able to produce Thr and Leu Thi not produced Resistant to S trepromycin

14. Media M9ST Agar Minimal Media 9 Streptomycin Antibiotic formation Inhibition of F- growth Thiamine Inhibition of F+ growth Promotion of HFR growth

15. Procedure Mix F- and HFR (F+) st rains Incubation in 37 o water bath for proper conjugation 30 minutes* Vortex to terminate genetic transfer Centrifugation – DNA Pellet formation Removal of supernatant (TSB broth )* Resuspension with M9 Broth Plated on M9ST Agar plates Incubation at 37 o for 48hrs

16. Results & Conclusion HFR ( F+) No growth Sensitivity to Antibiotics F - No Growth Absence of amino acids Mixed Culture Growth Produced Thr and Leu Antibiotic Resistance F- HFR

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

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