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

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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’

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 A G C T T 3’3’ T T C G A 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