Putting Microbes To Work In Biotechnology

Question 1

What is biological control

Answer 1

Use of biological agents for control of pests
Release of a small number of infectious agents that form a self-sustaining population in the pest species
Eradication vs controlling pest levels- economic threshold and economic injury level need to be monitored

Question 2

What things can insects be pests for

Answer 2

Agriculture
Pasture
Forestry
Medical and veterinary

Question 3

Insect pests

Answer 3

Transmit disease to plants= stress and damage plants
Significant damage to agricultural products intended for human foods and animal feeds

Question 4

Example of chemical treatment of manuka beetles in NZ and why its not used anymore

Answer 4

Specific to insects but can also affect humans
Effects the nervous system as it causes paralysis
Inhibits acetylcholinesterase which terminates signal transduction by getting rid of Ach that isnt used

Question 5

Biological management of pests

Answer 5

Physical removal
Use of resistant crops
Release of sterile males, natural enemies or pheromones
Microbial agents such as fungi, bacteria, viruses and nematodes

Question 6

What are entomopathogens and examples

Answer 6

Pathogens that kill or disable insects- highly specific target and facilitate the survival of beneficial insects in treated crops
Host provides optimal temperature and humidity
Bacteria: Serratia, Yersinia and Bacillus
Fungi: Cordyceps and Beauveria

Question 7

Serratia entomophila biocontrol

Answer 7

Grass grub specific, causes Amber disease (Amber colouration of larvae)
Carries disease specific plasmid= anti-feeding drugs
Uses nanodevice antifeeding prophage to deliver toxin Afp= cessation of feeding
SepABC insect active toxin complex leads to clearance of gut and amber colouration
Kills within 48 hr
Commercially available pesticide, no GMO

Question 8

Serratia proteamaculans biocontrol

Answer 8

AGR96X kills grass grub and makina beetle larvae
Leads to purple colouration
Kills in 5-12 days

Question 9

Yersinia entomophaga biocontrol

Answer 9

Against porina moth
Broad host range, safe for most insects
Non-toxic for birds and mammals
Toxin-bound chitinases degrade chitin rich peritrophic membrane of insect midgut
Receptor mediated endocytosis occurs
Toxin release either through; pore formation and translocation of toxic compounds, injection of toxin, dissolution of vesicle and toxin release
Leads to disintegration of midgut membrane, larvae vomit up their own intestines and die
Kills in 12hr-3days

Question 10

Bascillus thuringiemsis biocontrol

Answer 10

Produces insect toxins
Safe for non-target organisms, mammals cant activate toxin and other natural enemies arent affected= ecologically safe
Larvae ingest Bt toxin and activate it in the gut with high pH= pro-toxin
Protease activity cleaves pro-toxin into toxin which cleaves receptors in the gut
Binds with high affinity to endothelial gut, leads to pores and destruction of ion/ proton barrier
Influx of H2O= cell lysis
Insects die of infection and starvation within hours to weeks

Question 11

Genetically modified crops? Bt

Answer 11

Introduce the mechanisms of these bacteria/ the bacteria into the plants where they then become resistant themselves and there is no need for the bacteria to be sprayed in pesticides
Resistant crops and increases crop production
Safe for non-target organisms so can be eaten

Question 12

Fungi as biocontrol- Conidium

Answer 12

Pest (eg ant) takes up parasitic spore, penetrates cuticle, no injestion needed
Adheres to cuticle which determines fungal specificity
Spore germinates and forms germ tube with appressorium penetration structure- cuticle penetrated by mechanical pressure and cuticle degrading enzymes
Vegetative growth in host until reaches haemolymph where it spreads in blood
Starts to grow and kills ant from the inside out
Fungus still exposed to perfect conditions- temp, light, humidity

Question 13

Myco-insecticide fungal infection

Answer 13

Beauveria bassiana
Spores land on bugs, humidity and temp leads to germination of spores
Multiplication of fungus leads to release of toxins and draining of nutrients= death

Question 14

Parasitic wasps as biocontrol

Answer 14

Effective control of aphids
Senses distress signals of infested plants and honeydew sugars from aphid
Lands on and injects egg into aphid
Egg hatches and wasp grows, eating aphid from the inside out
Surviving aphids emit alarm pheromone causing colony to flee

Question 15

What are recombinant proteins

Answer 15

Protein encoded by recombinant DNA- coming from an artificial source not natural
Recombination (cloning), cloned in system that supports expression of gene, translation of mRNA and modification of DNA, purification and analysis

Question 16

What is a protein expression system and what needs to be considered with them

Answer 16

A living organism that is used to grow the desired protein
Most common= heterologous host; bacteria, yeast, algae, insect or mammalian
Need to think of cost, speed, PTMs eg glycosylation, folding and government regulation- all depend on what the protein is being used for and where it is coming from

Question 17

Applications of recombinant proteins

Answer 17

Biotherapeutics- vaccines eg HepB and TetanusC or hormones eg insulin, growth hormones, relaxin for induced labour
Food production- animal feed
Agriculture- crops
Bioengineering- enzymes, polymerases
Cytokines are also examples eg IL against microbes, IFN against viruses and anticancer to activate immune system

Question 18

Components of a simple cloning vector

Answer 18

Antibiotic resistance marker
Origin of replication
Multiple cloning site- has restriction sites for ligation and addition of genetic information
Constitutive promoter= not controlled

Question 19

Components of an expression vector

Answer 19

Inducible promoter- control expression
Shine delgarno sequence- for RNA pol binding
N or C terminal tag
Gene of interst
Transcription stop- dont want polymerase to keep going
PolyA site- optional

Question 20

Components of the lac operon

Answer 20

LacI repressor, binds to operator, removed by allolactose when lactose is present= control
Promoter and operator
LacZ= b-gal, cleaves lactose into glucose and galctose
LacY= b-gal permease, transport protein for lactose to enter cell
LacA= b-gal transacetylase, transfers acetyl groups from acetyl-CoA to b-gal, role not clear or known

Question 21

CAP- how it is activated

Answer 21

cAMP receptor protein
When glucose decreases, cAMP second messenger accumulates via intracellular signalling and activates CAP= increased gene expression

Question 22

How does CAP increase gene expression

Answer 22

Binds to major grooves in DNA upstream of RNA start site and opens it up allowing RNA pol to bind
Activates transcription through interaction with alpha subunit of CTD on RNA pol= enhanced efficiency

Question 23

IPTG and lac operon in the lab

Answer 23

Mimics allolactose= binds LacI and prevents binding to operator= constant expression
Not cleaved by B-gal like allolactose is= not stopped

Question 24

Protein affinity purification with a tag

Answer 24

GST, CBD and MBP are large tags not commonly used, his tag is used
Purified using column resin such as nickle that binds the tag
Protein elution with imidazole (histidine structural analogue) occurs
Can check with SDS-PAGE gel to see if we get our protein
Need to cleave off large tags (his is small so may not need to be cleaved) but this leaves some non-cleavable amino acids added to the protein structure

Question 25

Protein expression optimisation- vector stability

Answer 25

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)

Question 26

Protein expression optimisation- promoter activity

Answer 26

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

Question 27

T7 expression

Answer 27

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

Question 28

Improved tools for lac expression

Answer 28

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

Question 29

Protein expression optimisation- transcription terminators

Answer 29

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

Question 30

Protein expression optimisation- optimisation on plasmid

Answer 30

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

Question 31

Protein expression optimisation- translation initiation/ termination

Answer 31

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

Question 32

Protein expression optimisation- codon usage

Answer 32

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

Question 33

Protein expression optimisation- cellular compartment

Answer 33

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)

Question 34

Protein expression optimisation- proteolysis

Answer 34

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

Question 35

Protein expression optimisation- PTM

Answer 35

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

Question 36

Protein expression optimisation- large scale purification

Answer 36

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

Question 37

What are antibiotics

Answer 37

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

Question 38

Broad and narrow spectrum antibiotics

Answer 38

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

Question 39

Example of a bactericidal and bacteriostatic antibiotic

Answer 39

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

Question 40

How to find antibitoics

Answer 40

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

Question 41

Antibiotic targets

Answer 41

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

Question 42

Antibiotic resistance mechanisms

Answer 42

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

Question 43

Primary metabolites and antibiotics

Answer 43

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

Question 44

Secondary metabolites and antibiotics

Answer 44

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

Question 45

Isolation of active secondary metabolites

Answer 45

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

Question 46

Fungal secondary metabolites

Answer 46

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

Question 47

How to make penicillin

Answer 47

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

Question 48

Streptomyces and antibiotics

Answer 48

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

Question 49

Streptomyces coelicolor and antibiotics

Answer 49

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

Question 50

Limitations with secondary metabolites

Answer 50

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

Question 51

Plant secondary metabolites

Answer 51

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

Question 52

New antibiotics being produced and the issue

Answer 52

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