Term
| What are 5 traditional cell based screening methods? |
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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 |
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Term
| How do antimicrobial agents work? |
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Definition
| They penetrate the cell wall and disrupt key microbial functions |
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Term
| What are the 5 main antibacterial drug targets in bacteria and corresponding microbial agents? |
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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 |
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Term
| What are 5 mechanisms of genetic resistance to microbial agents? |
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Definition
1. decreased uptake
2. increased pump out
3. altered target site
4. enzymatic inactivation or modification
5. bypass pathways
6. overproduction of target |
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Term
| What are 5 examples of non- cell based new targets? |
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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 |
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Term
| what are 10 species that have already been shown to produce a high diversity of biologically active exometabolites? |
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Definition
1. aspergillus
2. bacillus
3. penicillium
4. daldinia
5. pseudomonas
6. alternaria
7. Streptomyces
8. actinomycetes
9. nocardia
10. fusarium |
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Term
| What are two methods that utilize the Ichip screening strategy to isolate unknown or "rare" microbes from ecological niches? |
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Definition
A. diffusion chamber
B. Microbial Trap |
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Term
| compare the 2 types of Ichip assemblies |
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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 |
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Term
| What culture conditions are needed to stimulate microbial production of secondary exometabolites from previously silent biosynthetic pathways? (6) |
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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 |
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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 |
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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 |
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Term
| selective isolation of ___ by pretreatment leads to new species and new ___ |
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Definition
| actinobacteria, exometabolites |
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Term
| What are 3 simple pretreatment isolation methods that would establish screeing bias? |
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Definition
heat pretreatment
chemical pretreatment
antibiotic pretreatment |
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Term
| Why are existing biomanufacturing strains used to screen for SBPs? |
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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) |
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Term
| What are 5 culture manipulation techniques conducted to screen for SBPs, in addition to the corresponding microbe used and compounds produced? |
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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 |
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Term
| culture manipulation often results in secretion of ___ |
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Definition
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Term
| describe 6 methods of co coultration of investigation sbps in microorgs and the corresponding outcomes |
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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 |
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Term
| What are the steps in genomic based and labeling approaches to screen for SBPs? |
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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 |
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Term
| Why are in vitro bioassays used? |
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Definition
| resistance sensitivity, tumor cell lines, activity screens, spectrum scrreens |
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Term
| compare and contrast in vitro and invivo assays |
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Definition
in vivo is animal models of infections, cancers
in vitro is tumor cell lines, ativity screens |
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Term
| why use agar diffusion bioasssays or enzyme assays during process modeling? |
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Definition
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Term
| in growth inhibition assays the degree of growth inhibition is related to __- an estimated concentration also known as the ___ |
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Definition
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