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Transcript
Antibiotics Ampicillin and Kanamycin Ampicillin -Inihibits cell wall synthesis -Irreversible inhibits transpeptidase, which cross-links the peptidoglycan 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 Kanamycin -an aminoglycoside (amino modified sugar) bacteriocidal antibiotic -Isolated from Streptomyces kanamyceticus -Binds to the 30S procaryotic ribosome Kanamycin -Binds to the 30S procaryotic ribosome Kanamycin Resistance -aminoglycoside modifying enzymes, phosphorylation, making Kanamycin uneffective Bacteriostat vs. Bacteriocide Ampicillin – inhibits new cell wall synthesis Kanamycin – inhibits protein synthesis 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 DNA Properties ?????? 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’ 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 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 3’ T T C G A A G C T T 3’ A 5’ Lambda (λ) Phage -Bacteriophage with double-stranded DNA genome, 48,502 bp DNA Gel Electrophoresis -Separation of DNA fragments through agarose by application of an electric field DNA Gel Electrophoresis DNA Gel Electrophoresis -DNA Mobility is inversely proportional to Log10 of its Molecular Weight
