Putting Microbes To Work In Biotechnology Flashcards

Question

Protein expression optimisation- vector stability

Answer 1

Host specificity- organism needs to be able to recognise ori Type of vector eg cloning, broad host (own replication gene so recognises own ori), shuttle (two ori so ensures specific organisms will recognise), reporter (has something like a fluorescent protein that can be studied for efficacy) Plasmid copy number by origin of replication, size of plasmid or insert (is it a metabolic burden)

Answer 2

Inducibility level- strong but not too strong Easily inducible- positive or negative Promoter optimisation- different strengths Add with fluorescent protein to see how different promoters affect strength of expression

Answer 3

Expression vector into E.coli T7 expression host Use bacteriophage T7 RNA pol which has high affinity for T7 promoter sequence= very specific and more yield

Answer 4

UV5 promoter Edits CAP binding site so insensitive to CAP and removes cAMP regulation

Answer 5

Intrinsic terminator/ Rho independent which uses RNA encoded stem-loop Rho dependent which is where there is a sequence that slows or stops RNA pol and the Rho protein following bumps into it and they dissociate Need to know what control the organism is under

Answer 6

Transcripts into replication region destabilises plasmids- terminators enhance stability Transcript through a promoter affects expression

Answer 7

Initiation: ribosomes scan mRNA for shine delgarno sequence- can be different in different organisms so need one able to be recognised, distance between ribosome binding site and ATG influences expression Termination: Stop codon, more efficient if followed by another T as it affects rate ribosome falls off

Answer 8

Rare codons= low tRNA levels to regulate protein levels Codon usage bias- where a codon coding for something in humans may have less chance of coding for the same thing in another organism- frequency table for optimal expression needs to be considered

Answer 9

Important it is known where protein of interest goes: Cytoplasm, periplasm or extracellular (requiring signal peptide) Cells can have inclusion bodies filled with proteins they dont know what to do with and cant degrade quick enough Could lyse cells and get inclusion bodies and get proteins out (often denatured inside so need to make back into correct shape)

Answer 10

Common in bacteria Problem as it can cleave protein of interest Optimisation: knockout E.coli BL21 and Lon which degrade certain proteins, removal of protease cleavage sites and fusion proteins

Answer 11

Mainly needed in mammalian proteins Done to improve solubility and stability Attachment of a molecules to a functional group of another molecule; glycosylation, hydroxylation, phosphorylation, acetylation or methylation

Answer 12

Optimise expression strain development Fermentation allows control of physical parameters- temp, O2, pH, nutrient levels Need to get rid of and control biological waste/ solvent

Answer 13

Chemicals that kill (bactericidal) or inhibit (bacteriostatic) growth of bacteria, used to treat bacterial infection Produced in nature by soil bacteria and fungi Natural defence against other microbes produced by microbes in stress situations

Answer 14

Broad= act against a large group of bacteria- can test and get better treatment once given to patient- hope that something in there targets what they have as initially dont know what someone has Narrow= act against a limited group of bacteria- specific and need to know what the pathogen is to use these= limitation

Answer 15

Bactericidal= B-lactams eg penicillin, works to inhibit cell wall biosynthesis Becteriostatic= sulfonamides eg prontosil, works to prevent growth and replication and causes allergic reactions in some patients

Answer 16

Collect soil/ environment sample Grow microbes on a plate Loos for inhibition zones against other indicator strains eg E.coli whcih are common Isolate a pure culture Test activity against many strains Isolate active ingredient if this can be done Characterise via chemical stability and toxicity

Answer 17

Cell wall or membranes Machinery that make nucleic acids- DNA/RNA Machinery that produce protein- ribosome and associated proteins

Answer 18

Porins- reduce the number and reduce cell permeability for drugs to enter Efflux pumps- suck out drugs that cross the periplasm Target site modification- introduction of mutations Antibiotic resistance genes acquired through HGT Antibiotic modifying enzymes- modifies antibiotic so it cant bind

Answer 19

Produced by all microbes for growth Produced during active growth in lag, exponential and stationary phase Eg lactic acid, amino acids, proteins, enzymes, nucleic acid, ethanol No pharmaceutical benefits/ no antimicrobial activity Stimulate production of secondary metabolites= introduction of biosynthetic enzymes and increase amount of precursors

Answer 20

Biologically active small molecules Made when bacteria under stress, not required for viability Need precursors- normally primary metabolites Competitive weapons against other bacteria and fungi such as toxin production

Answer 21

Production starts in stationary phase- lack of nutrients, environmental stress, not essential for growth Ecological role important- can have many roles Products can be toxic to mammals eg exotoxin

Answer 22

Protein producers- Claviceps purpurea Grows on ears and rye and relates cereal plants Production of poisonous alkaloids- ergometrine Ergotism or death when ingested= convulsive (seizures) and gangrene (tissue death) symptoms Antibiotic producers of penicillin and cephalosporins

Answer 23

From fungus Penicillum Fermentation- allows cotnrol of conditions Vessel filled with medium containing sugars (for respiration) and ammonium salts (for proteins and nucleic acids) Penicillum added When sugar goes below certain level leads to secondary metabolite penicillin production

Answer 24

Complex secondary metabolism Synthesis naturally produced antibiotics Make >100,000 bioactive compounds and produce 2/3 of clinically used antibiotics Antifungal, antiparasitic immunosuppressants

Answer 25

Best studied organism for secondary metabolism Produces 2 pigmented secondary metabolites- blu/ green in alkali and red in acidic Most well understood secondary metablome Biosynthesis of pigmented antibiotics Actinorhodin antibiotic is aromatic polyketide, enzyme encoded in 22-kb gene cluster

Answer 26

Many are expressed at low levels during growth in the lab Expression of biosynthetic gene clusters (multiple genes) means need to understand how everything is connected and pathways Limited precursor availability Regulatory network that controls expression of metabolic genes is complex and largely unknown Even if new antibiotics are found, there may already be resistance out there against them= issue in itself

Answer 27

Pharmacological active natural compounds Medical treatments Anticancer- paclitaxel, vinblastine Antimalaria- artemisinin Opioid analgesic drug- morphine Alzheimers- galantamine Unsure how primary feeds into secondary- very complex

Answer 28

Less than 1% of antimicrobial agents have medical or commercial value Means the new ones being found are not of any use