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

The mechanism of antibiotics
Biol 1220 Synthetic Biology
abe pressman & minoo ramanathan
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
sites of antiobiotic action
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?
ampicillin resistance
• Cleavage of β-lactam ring by β-lactamase
enzyme
ampicillin resistance
• β-lactamase is encoded by the plasmid-linked
bla (TEM-1) gene
• Hydrolyzes ampicillin
• Ampicillin levels in culture continually
depleted
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
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
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
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
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://abe.leeward.hawaii.edu/Protocols/QiagenSpinprepProtocol.htm
• http://www.openwetware.org/wiki/Brown_BIOL1220:Notebook/SynBio_i
n_Theory_and_Practice/Bacterial_Basics