2. ANTIBIOTICS
A chemical compound acting against life
is known as antibiotics. It can obtain
either from natural sources ,microbes or
by synthetic methods.
4. 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
7. PENICILLIN
most widely used antibiotic.
It is drug of choice when infection is caused by
organisms susceptible to it .
It is effective drug
against gram + bacteria
and also against rickettsia .
Mostly penicillin are
produced by species of
penicillium .
8. PENICILLIN BIOSYNTHESIS
Overall, there are three main and important steps to the biosynthesis of penicillin
G (benzylpenicillin).
9. FIRST STEP
is the condensation of three amino acids — L-α-
aminoadipic acid, L-cysteine, L-valine into
a tripeptide. Before condensing into the tripeptide, the
amino acid L-valine must undergo epimerization to
become D-valine. The condensed tripeptide is named
δ-(L-α-aminoadipyl)-L-cysteine-D-valine (ACV). The
condensation reaction and epimerization are both
catalyzed by the enzyme δ-(L-α-aminoadipyl)-L-
cysteine-D-valine synthetase (ACVS), a nonribosomal
peptide synthetase or NRPS.
10. SECOND STEP
The second step in the biosynthesis of
penicillin G is the oxidative conversion
of linear ACV into
the bicyclic intermediate isopenicillin N
by isopenicillin N synthase (IPNS),
which is encoded by the
gene pcbC. Isopenicillin N is a very weak
intermediate, because it does not show
strong antibiotic activity.
11. FINAL STEP
is an transamidation by isopenicillin N
N-acyltransferase, in which the α-
aminoadipyl side-chain of isopenicillin
N is removed and exchanged for
a phenylacetyl side-chain. This
reaction is encoded by the
gene penDE, which is unique in the
process of obtaining penicillins
12. PRODUCTION
Penicillin is a secondary metabolite of certain species of Penicillium and is produced
when growth of the fungus is inhibited by stress. It is not produced during active
growth. Production is also limited by feedback in the synthesis pathway of penicillin.
α-ketoglutarate + AcCoA → homocitrate → L-α-aminoadipic acid → L-lysine + β-
lactam The by-product, L-lysine, inhibits the production of homocitrate, so the
presence of exogenous lysine should be avoided in penicillin production.
The Penicillium cells are grown using a technique called fed-batch culture, in which
the cells are constantly subject to stress, which is required for induction of penicillin
production. The available carbon sources are also important: Glucose inhibits
penicillin production, whereas lactose does not. The pH and the levels of nitrogen,
lysine, phosphate, and oxygen of the batches must also be carefully controlled.
The biotechnological method of directed evolution has been applied to produce by
mutation a large number of Penicillium strains. These techniques includeerror-prone
PCR, DNA shuffling, ITCHY, and strand-overlap PCR.
Semisynthetic penicillins are prepared starting from the penicillin nucleus 6-APA.
13. STREPTOMYCIN
It is produced using strains of streptomyces griseus . Basic
medium for production of sterptomycin contains
soybean meal as nitrogen source ,
glucose as carbon source and NaCl .
Proteolytic enzymatic activity of
S.griseus releases ammonia to the
medium from the soybean meal
causing rise in pH .during this initial
fermentation phase there is a little
production of streptomycin.
15. A little additional production of mycelia. The glucose added in the
medium & the ammonia released from the soybean meal are
consumed during this phase. The Ph remains fairly constant (7.6- 8).
It is the final phase of the fermentation, after depletion of
carbohydrates from the
medium, Streptomycin production
ceases & the bacterial cells began to
lyse. There is a rapid increase in ph
because of the release of ammonia from
lysed cells. In the end of fermentation, the
mycleium is separated from the broth
by filtration & the streptomycin is recovered. The purification consists
of adsorbing the streptomycin onto activated charcoal & eluting with
acid alcohol.
16. 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
18. β-lactamase is encoded by the
plasmid-linked bla (TEM-1)
gene
Hydrolyzes ampicillin
Ampicillin levels in culture
continually depleted
19. 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
20. 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
21. 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.
22. 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
An antibiotic is a substance or compound that that kills or inhibits the growth of bacteria. They are broadly classified based on lab behavior as bactericidal (kill bacteria directly) or bacteriostatic (prevent cell division).Also categorized by target-specificity: Narrow-spectrum antibiotics target specific bacteria such as Gram-positive or Gram-negative bacteria, while broad-spectrum antibiotics affect a wider range of bacteria.
This diagram shows the various targets of antibiotics on the structure and functions carried out by bacteria. The beta-lactams group of antibiotics, which I will talk more about shortly, has an effect on the construction of cell walls by many different types of bacteria.
Ampicillin is a type of beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. It is considered part of the aminopenicillin family and belongs to the B-lactam group of antibiotics because of its characteristic B-lactam ring that consists of 3 carbon atoms and 1 nitrogren atom. (Show Slide 4)Ampicillin is a broad-range antibiotic so it can penetrate both Gram-positive and Gram-negative bacteria. The penicillin derivative acts as a competitive inhibitor of the enzyme transpeptidase, found on the inner surface of the cell surface membrane and which is essential for bacterial cell wall synthesis. It inhibits the third and final stage of cell wall synthesis, which ultimately leads to cell lysis.Ampicillin can also act as a bactericidal in the presence of E. Coli bacteria.However, as I mentioned before, beta-lactam drugs such as ampicillin have been popular for decades for the treatment of bacterial infection, hence certain bacteria have been able to develop counter-measures to nullify the antibiotics effectiveness. This evolutionary process is known as a buildup of drug resistance. There are several different underlying molecular mechanisms to antibiotic resistance, and I will explain the mechanism specific to ampicillin.
Ampicillin resistance is achieved by the cleavage of the beta-lactam ring by the beta-lactamase enzyme.
The enzyme is coded for by the plasmid-linked bla gene which hydrolyzes ampicillin by cleaving the beta-lactam ring. This can be witnessed when a plasmid that has been inserted into a bacterium is first coupled with the bla gene and then the bacterium is placed in a culture medium containing ampicillin – causing ampicillin levels to be continually depleted.
So now the most important question, is how the ampicillin-beta lactamase mechanism can be useful to us as a synthetic biology technique?It is often used as a selective agent to confirm the uptake of genes by bacteria (eg. plasmids). As we heard in a previous presentation, bacterial transformation results in the integration of the same of foreign DNA from the media surrounding the bacteria to produce chemically competent cells.If the exogenous DNA is tagged with an antibiotic resistance gene eg beta-lactamase and then grown in a medium containing ampicillin, only the bacteria that had successfully taken up the desired DNA become ampicillin resistant and do not become lysed by the ampicillin. This is then quite an accurate way to confirm whether or not successful bacterial transformation has occurred.
Kanamycin is a chemical compound which targets the 30s ribosomal subunit in prokaryotes, binding in such a way as to cause a frameshift in every translation. This has a "bacteriostatic" effect: the bacterium is unable to produce any proteins correctly, leading to a halt in growth and eventually cell death.
Over-use of kanamycin has led to many wild bacteria possessing resistance, which is encoded in 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 comes in three variaties: antibiotic-degrading enzymes, reduced membrane permeability, or proteins protecting the 30s subunit.
Chloroamphenicol is also bacterio static; it halts bacterial growth by inhibiting the enzyme peptidyl transferase, a protein that assists in the binding of tRNA to the 50s ribosomal subunit in prokaryotes.Three methods of resistance exist: reduced membrane permeability, mutation of the 50s subunit, and an enzyme called chloramphenicol acetyltransferase, which inactivates chloramphenicol by covaltly linking groups.Indeed, there is much similarity in function, use and resistance between kanamycin and Chloroamphenicol.