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Antibiotics

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  1. Antibiotics

  2. Ampicillin and Kanamycin

  3. Ampicillin -Inihibits cell wall synthesis

  4. -Irreversible inhibits transpeptidase, which cross-links the peptidoglycan

  5. Ampicillin Resistance -beta-lactam antibiotic -Acquires a gene that encodes β-lactamase -Cleaves β-lactam ring of ampicillin -Ampicillin is no longer bound to transpeptidase and therefore functions normally

  6. Kanamycin -an aminoglycoside (amino modified sugar) bacteriocidal antibiotic -Isolated from Streptomyces kanamyceticus -Binds to the 30S procaryotic ribosome

  7. Kanamycin -Binds to the 30S procaryotic ribosome

  8. Kanamycin Resistance -aminoglycoside modifying enzymes, phosphorylation, making Kanamycin uneffective

  9. Bacteriostat vs. Bacteriocide Ampicillin – inhibits new cell wall synthesis Kanamycin – inhibits protein synthesis

  10. Restriction Endonucleases Nuclease – Enzymes that digest or cut DNA and RNA Endonuclease vs. Exonuclease Endonuclease – Cut nucleic acids at sites internal to the molecule Exonuclease - Cut nucleic acids at sites external to the molecule

  11. DNA Properties ??????

  12. Restriction Endonuclease -1950s, a “primitive immune system” in E. coli in defense of bacteriophage lambda infection -1968, Restriction enzyme HindIII was isolated from Haemophilus influenzae -Recognition site for HindIII 5’ G T A C 3’ 3’ C A T G 5’

  13. Restriction Endonuclease -1978, Nobel prize awards for the discovery of restriction endonucleases and their application to molecular genetics -Currently over 3000 restriction endonulceases isolated with 230 recognition sequences -Mostly isolated from bacteria, but some from viruses, archaea, and eucaryotes

  14. HindII -Recognition site for HindIII 5’ A A G C T T 3’ 3’ T T C G A A 5’ -Cleavage Products 5’ A A G C T T 3’ 3’ T T C G A A 5’

  15. Lambda (λ) Phage -Bacteriophage with double-stranded DNA genome, 48,502 bp

  16. DNA Gel Electrophoresis -Separation of DNA fragments through agarose by application of an electric field

  17. DNA Gel Electrophoresis

  18. DNA Gel Electrophoresis -DNA Mobility is inversely proportional to Log10 of its Molecular Weight