test and quiz 2 – Flashcards
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| list the stable expression of recombinant proteins in mammalian cell line expression systesm (5) |
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| 1. heterologous DNA is integrated into the host genome and is maintained throughout many generations 2. cell line development requires an extensive period (6-12 months) 3. after amplification and high producer selection, expression level is relatively high 4. stable cell lines can be used for large scale manufacturing leading to clinical trials and commercialization 5. high cost |
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| agar diffusion bioassay |
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| classical biological activity assay used for initial screening of the biological activity of UNPURIFIED compounds particularly for bioactive compounds produced by strains isolated from nature. A nonsterile preparation of the test material is deposited on a paper disk placed on a petri dish containing agar pre-inoculated with a reference or test organisms susceptible to growth inhibition by the action of one or more compounds in the sample. The compounds diffuse into the agar from the paper disk during incubation. as the test organism is the agar grows a grwoth inhibition zone is generated around the disk if the compound has antimicrobial activity for the test organism. the square of the size of the inhibition zone is proprotional to the titer of the compound in the preparation |
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| NRPS |
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| a peptide producing enzyme complex which assembles amino acids independent of messenger RNA. Each NRPS can produce one type of non ribsomal peptide which usually features cyclic or highly branched structures some of these structures have antibacterial properties and are commercial products |
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| BEVS |
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| baculovirus expression vector system: insect cells expression system based on the introduction of the target gene into a nonessnetial bacuovirus replication genome region by homologous recombination or transposition expression can be made by infecting insect cells or larvae and allowing the virus to replicate reaching very high virus and recombinant protein yields due to the strong virus promoter |
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| Glutamine synthase expression system |
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| it is the method of choice for selection of highly productive mammalian cells many strains require glutamine so by using a vector containing the target gene and the GS gene only recombinant cells can survive in glutamine free media further improvement of protein expression can be accomplished by using a weak promoter for the GS gene and a strong promoter for the target gene |
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| pks |
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| polyketide synthase this is a family of enzyme complexes coded by genes that are usually organized in a single operon in bacterial genomes. PKS can take the form of a large multi domain enzyme or an aggregate of monofunctional simple proteins PKS are important because they produce many secondary metabolites with antimicrobial activity in bacteria and fungi the characteristic mechanism that yields these compounds is commonly compared to an assembly line where the precursor molecule moves from one reactive site to another as it grows and adds more functional groups |
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| antibiotic resistance genes |
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| genes that code for molecules that mediate cellular resistance to antibiotices these mechanisms include: beta lactamase production genes coding for mutated penicillin binding proteins with decreased affinity for beta lactam binding down regulation of porin channels for liminting the entry of antibiotic into the periplasm |
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| what are the steps used to express and purify recombinant proteins using engineered baculovirus system on the industrial scale? |
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| 1. transfection into insect cells with recombinant baculoviral dna 2. amplification of viral stock in adherent cultures 3. protein expression in suspension cultures 4. protein purificatoin 5. scaling up protein expression for industrial production |
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| what are the advantages of using fungi compared to E.coli? (5) |
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| 1. gras 2. excellent enzyme secretors 3. large scale cultivation procedures 4. no excesseive hyper mannosylation 5. very high production yields |
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| what are the disadvantages of protein expression using filamentous fungi compared to e. coli |
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| 1. heavily patented 2. difficult genetic manipulation 3. abundant production of endogenous and extracellular protease 4. potential toxin production |
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| cultivation conditions have been shown to alter the secondary metabolites secreted from antibiotic producting microbes. This is called activation of silent biosynthetic pathways. explain how altering the feeding of carbon, nitrogen, or phosphate sources in a growth medium of a strain of streptomyces can alter spb enzyme expression |
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| the carbon, nitrogen, phosphate and trace metal concentrations and sources must be chosen in such a way that transcription of antibiotic biosynthethtic genes is not repressed. slow feeding of highly soluble sources of c, n, phosphate are difficult to control as nutrients are rapidly absorbed. changes in any of these elements can also produce undesirbale batch to batch variablility or complete production repression. |
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| outline how you would design a screening process to find a soil microbe that produces a novel (patentable)compound that is similar to beta lactams and can be taken orally and resistant to beta lactamase cleave |
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| a. primary screen: impose soil screening bias by pre-treatment with heat and chlorine, screen different soil depths b. select streptomyces by culture on chitin media at low pH and 50 c (stomach conditions) c. search for slow growth (2-3 weeks and complex nutritional requirements d. in vitro bioassay for biological activity using e. coli as a model of human pathogenic bacteria e. extraction and purification of novel antibacterial compounds for rapid dereplication to determine if novel structures related to clavulanic acid are formed that may be patentable f. confirm bioactivity of purified compounds w/weakly human pathogenic bacterial strains in vitro and in vivo with a model bacterial infection in rodents. |
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| a precursor fed or "directed fermentation process |
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| adding a desired side chain or precursor building block to an antibiotic producing organisms culture medium to get this building block incorporated into the final secreted antibiotic molecule |
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| space time yield |
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| represents the mass or product formed per volume of react and time |
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| alginate hydrogel beads |
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| highly porous matrix formed by divalent cation cross linking of L-glutornic acid. relatively weak but characterized by high diffusion coefficients |
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| clavulanic acid |
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| beta lactamase inhibitor similar in structure to penicillins that is used to overcome antibiotic resistance in bacteria that produce b lactamase which inactivates most penicillins. It is weakly antibiotic and is produced by streptomyces clavurigerus |
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| what are 4 different types of biocatalyst immobilization methods |
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| 1. surface attachment 2. entrapment within porous matrices 3. containment behind a barrier 4. self aggregation can all be used for microbial cells cross linking can result in cell death |
