1. The mechanism of antibiotics Biol 1220 Synthetic Biology abe pressman & minoo ramanathan

2. the basics Used to kill or inhibit the growth of bacteria Classified as bactericidal or bacteriostatic Kill bacteria directly Prevent cell division Classified by target specificity: Narrow-spectrum vs Broad range Most modified chemically from original compounds found in nature, some isolated and produced from living organisms

3. sites of antiobiotic action

4. ampicillin Belongs to β-lactam group of antibiotics – contain β-lactam ring Broad-spectrum Penicillin derivative that inhibits bacterial cell wall synthesis (peptidoglycan cross-linking) Inactivates transpeptidases on the inner surface of the bacterial cell membrane Bactericidal only to growing E. Coli Widespread use leads to bacterial resistance. HOW?

5. ampicillin resistance Cleavage of β-lactam ring by β-lactamase enzyme

6. ampicillin resistance β-lactamase is encoded by the plasmid-linked bla (TEM-1) gene Hydrolyzes ampicillin Ampicillin levels in culture continually depleted

7. use in synthetic biology To confirm uptake of gene (eg. of plasmids) by bacteria Bacterial Transformation: DNA integrates into bacteria’s chromosome and made chemically competent Exogenous DNA tagged with an antibiotic resistance gene eg. β-lactamase Grown in medium containing ampicillin Ampicillin resistance indicates successful bacterial transformation

8. Kanamycin Targets 30s ribosomal subunit, causing a frameshift in every translation Bacteriostatic: bacterium is unable to produce any proteins correctly, leading to a halt in growth and eventually cell death

9. kanamycin use/resistance Over-use of kanamycin has led to many wild bacteria possessing resistance plasmids As a result of this (as well as a lot of side effects in humans), kanamycin is widely used for genetic purposes rather than medicinal purposes, especially in transgenic plants Resistance is often to a family of related antibiotics, and can include antibiotic-degrading enzymes or proteins protecting the 30s subunit

10. chloramphenicol Bacteriostatic: functions by halting bacterial growth, which is done by inhibiting the enzyme peptidyl transferase, a protein that assists in the binding of tRNA to the 50s ribosomal subunit Three methods of resistance: reduced membrane permeability, mutation of the 50s subunit, and an enzyme called chloramphenicol acetyltransferase, which inactivates chloramphenicol by covaltly linking groups Easy/cheap to manufacture, but unused in western countries because of possible aplastic anemia as a side effect

11. Sources http://en.wikipedia.org/wiki/Antibiotic http://en.wikipedia.org/wiki/Ampicillin http://en.wikipedia.org/wiki/Beta-lactamase http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=A1593|SIAL &N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=A1593|SIAL &N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC http://abe.leeward.hawaii.edu/Protocols/QiagenSpinprepProtocol.htm http://www.openwetware.org/wiki/Brown_BIOL1220:Notebook/SynBio_i n_Theory_and_Practice/Bacterial_Basics http://www.openwetware.org/wiki/Brown_BIOL1220:Notebook/SynBio_i n_Theory_and_Practice/Bacterial_Basics