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Antibiotics
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Ampicillin and Kanamycin
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Ampicillin -Inihibits cell wall synthesis
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-Irreversible inhibits transpeptidase, which cross-links
the peptidoglycan
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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
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Kanamycin -an aminoglycoside (amino modified sugar) bacteriocidal antibiotic -Isolated from Streptomyces kanamyceticus -Binds to the 30S procaryotic ribosome
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Kanamycin -Binds to the 30S procaryotic ribosome
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Kanamycin Resistance -aminoglycoside modifying enzymes, phosphorylation, making Kanamycin uneffective
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Bacteriostat vs. Bacteriocide
Ampicillin – inhibits new cell wall synthesis Kanamycin – inhibits protein synthesis
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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
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DNA Properties ??????
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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’
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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
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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’
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Lambda (λ) Phage -Bacteriophage with double-stranded DNA genome, 48,502 bp
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DNA Gel Electrophoresis
-Separation of DNA fragments through agarose by application of an electric field
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DNA Gel Electrophoresis
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DNA Gel Electrophoresis
-DNA Mobility is inversely proportional to Log10 of its Molecular Weight
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