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lecture 7
lecture 7
21
Chemical Engineering
Post-Graduate
10/23/2013

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Term
What are 5 traditional cell based screening methods?
Definition
Primary Screens/pre screening basis
reporter, antisense, reversion assays on a micro scale
biological activity screens
correlative bioassays
extraction, separation of exometabolites
dereplication of "hits"
toxicity testing in animals
in vitro efficacy testing in animal models
Term
How do antimicrobial agents work?
Definition
They penetrate the cell wall and disrupt key microbial functions
Term
What are the 5 main antibacterial drug targets in bacteria and corresponding microbial agents?
Definition
1. Cell wall synthesis (penicillins, cephalosporins, carbapenems)
2. folic acid metabolism (sulphonamides
3. protein synthesis (2- 508 inhibitors macrolides, 305 inhibitors aminoglycosides, tetracyclines
4. DNA synthesis quinolones
Term
What are 5 mechanisms of genetic resistance to microbial agents?
Definition
1. decreased uptake
2. increased pump out
3. altered target site
4. enzymatic inactivation or modification
5. bypass pathways
6. overproduction of target
Term
What are 5 examples of non- cell based new targets?
Definition
1. cell wall biosynthesis inhibitors
2. transglycosylase inhibitors
3. new protein synthesis inhibitors
4. reverse transcriptase, protease inhibitors
5. efflux blockers
6. tubulin, microtubule, actin binding
7. cell surface receptors, nuclear receptors
Term
what are 10 species that have already been shown to produce a high diversity of biologically active exometabolites?
Definition
1. aspergillus
2. bacillus
3. penicillium
4. daldinia
5. pseudomonas
6. alternaria
7. Streptomyces
8. actinomycetes
9. nocardia
10. fusarium
Term
What are two methods that utilize the Ichip screening strategy to isolate unknown or "rare" microbes from ecological niches?
Definition
A. diffusion chamber
B. Microbial Trap
Term
compare the 2 types of Ichip assemblies
Definition
Diffusion chamber has a pre-inoculated agar, and the membrane prevents entry of bacteria
B. microbial trap agar matrix not preinoculated, the 0.2 micron membrane allows entry of actinomycetes
Term
What culture conditions are needed to stimulate microbial production of secondary exometabolites from previously silent biosynthetic pathways? (6)
Definition
1. liquid vs. solid agar (plugs)
2. trace metals, potassium, mg, sulfate, P, N-source, C-sources
3. limit carbon sources: glucose, starch, cellulose, plant extracts (malt, potato, carrot)
4. no single medium can support secondary exometabolite in a variety of species (multiple media needed)
5. temp, redox, ph, water activity optimal for growth does not equal optimal for secondary exometabolite production
6. slow growth - 5-10 day biomanufacturing process
Term
Slow growing mycelial soli bacteria such as actiomycetes and fungi such as penicillium produce many types of antibiotics. screening strategies to find new compounds should avoid re-isolating these same strains. What steps should be followed to isolate new or "rare" strains of actinomycetes
Definition
soil pretreated with antibiotics, organic solvents
different soil depths
selection on chitin media (high pH, growth at 50 C)
search for slow growth (2-3 weeks) complex nutritional requirement, motile zoospores
Term
selective isolation of ___ by pretreatment leads to new species and new ___
Definition
actinobacteria, exometabolites
Term
What are 3 simple pretreatment isolation methods that would establish screeing bias?
Definition
heat pretreatment
chemical pretreatment
antibiotic pretreatment
Term
Why are existing biomanufacturing strains used to screen for SBPs?
Definition
1. microbes express secondary exometabolite pathways under lab conditions
2. abiotic vs biotic factors important in biosynthesis of secondary exometabolites
3. culture conditions are important
4. co cultivation methods used (bacteria and fungi) to detect signaling molecules
addition of inhibitors (non-specific, specific)
Term
What are 5 culture manipulation techniques conducted to screen for SBPs, in addition to the corresponding microbe used and compounds produced?
Definition
aspergillus ochraceu- variation of media composition, vessel, and oxygenation (total of 15 compounds produced)
chaetomium chiversii (switch from solid to liquid medium)
carbon source of media varied
addition of scandium and other rare elements
shifting temperatures
addition of dimethyl sulfoxide
Term
culture manipulation often results in secretion of ___
Definition
new compounds
Term
describe 6 methods of co coultration of investigation sbps in microorgs and the corresponding outcomes
Definition
1. marine bacteria cocultured with live and heat killed terrestrial bacterial (produced uncharacterized antibiotics)
2. marine bacteria cocultured with live and/or cell free supernatents of marine and terrestrial bacterial (same as above
2 live fungi cocultured together
induced production of acremostatins
Term
What are the steps in genomic based and labeling approaches to screen for SBPs?
Definition
1. scan for SBPs
2. predict new structure based on genome sequence
3. heterologous expression of SBPs
4. few standard SBP genetic methods
5. chemical epigenitc methods
6. genomisotrohic- combine gene cluster+ structure prediction + isotopic labeling by precursor feeding
Term
Why are in vitro bioassays used?
Definition
resistance sensitivity, tumor cell lines, activity screens, spectrum scrreens
Term
compare and contrast in vitro and invivo assays
Definition
in vivo is animal models of infections, cancers
in vitro is tumor cell lines, ativity screens
Term
why use agar diffusion bioasssays or enzyme assays during process modeling?
Definition
rapid, inexpensive
Term
in growth inhibition assays the degree of growth inhibition is related to __- an estimated concentration also known as the ___
Definition
titer, dilution unit
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