1. Antibiotics

2. Ampicillin and Kanamycin

3. Ampicillin
-Inihibits cell wall synthesis

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

6. 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

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

8. Kanamycin
-Binds to the 30S procaryotic ribosome

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

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

11. 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

12. DNA Properties
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13. 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’

14. 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

15. 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’

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

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

18. DNA Gel Electrophoresis

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