Module 5 - Exploiting Microorganisms Flashcards
Describe the difference between transgenic and cisgenic
Transgenic = genetic material has been transferred between unalike species
Cisgenic = gene has been edited within a single species
Name the genus of bacteria that cause Crown Gall Disease
Agrobacterium
What triggers Agrobacterium infection of a plant?
When a plant is wounded it releases Acetosyringone - the VirA/G protein products detect this molecule and the Agro moves via chemotaxis towards it
What does virD2 do following activation by VirA/G?
It excises the T-DNA from the Ti plasmid and transports it into the plant cell
What do virD4 and virB do in Agro infection?
They connect the Agro cell to the plant cell
What does LRR do in the plant defence system?
It recognises the flagellum and activates a signalling cascade
Describe the signalling cascade when LRR is activated
Phosphorylation of MKK4/5 -> Phosphorylation of VIP1 to VIP1p -> VIP1p enters the nucleus and activates transcription of genes encoding antipathogenic response
What does VirF do in Agro infection?
VirF degrades VIP1, preventing antipathogenic genes from being transcribed
What does VirE2 do in Agro infection?
VirE2 coats the T-DNA as VirD2 transports it into the nucleus.
VirE2 also binds VIP1, thus using the plant’s own defence mechanism for its own gain
Once T-DNA is integrated into the plant genome, it promotes the synthesis of which 3 proteins?
Auxin, cytokinin and opines
Which 2 genes are removed from the T-DNA region in genetic modification?
Genes for hormone and opine biosynthesis
Which 2 genes are inserted into T-DNA in genetic modification?
The Gene of Interest, and an antibiotic resistance gene
Why are binary vector systems necessary in genetic engineering using T-DNA?
EcoRI (restriction enzyme) has a recognition site of 6 bases (once every 4000), so would cut the Ti plasmid into many inconvenient fragments
What is the first step in transforming plant cells with T-DNA?
Co-Cultivation (leaf discs placed on agar plate; solution of Agro carrying binary vector poured on top)
What is the second step in transforming plant cells with T-DNA?
Selection using selectable antibiotic marker (e.g., kanamycin)
What is the third step in transforming plant cells with T-DNA?
Regeneration:
Many plant cells are totipotent -> manipulate cell division using endogenous plant growth regulators (e.g., auxin, cytokinin) -> cells will organise themselves to form shoot meristem
What part of the plant is the target for transformation using flower dipping methods?
Ovules (in the flowers)
Name the species responsible for the Bt toxin
Bacillus thuringiensis
Name the three orders of insects affected by Bt toxin
Lepidoptera (Moths/Butterflies), Diptera (Flies), Coleoptera (Beetles)
Why does the Bt toxin only affect insects?
It is insoluble under normal conditions, but under the alkaline (high pH) conditions of the insect mid-gut, the protoxin is proteolytically cleaved to give active, soluble toxin
How useful is dried and sprayed Bt as a direct insecticide?
Not very - unstable and only provides protection for a few days
How is Bt toxin made more stable for use on cotton plants?
Cloned into another bacterium - Pseudomonas flourescens
How can the Bt toxin be expressed in plants themselves?
Place the gene in front of the 35S promoter to transform the plant; if larvae feed on plant they ingest the toxin
Which biosynthetic pathways are generally targeted by herbicides?
Vitamin synthesis and amino acid synthesis
State 5 problems with conventional herbicides
- Hard to distinguish very similar species
- Often have poor environmental properties
- May need more than one type of herbicide
- When used as pre-emergence sprays they can be wasteful
- Resistance is a growing problem
What is the name of the pathway that glyphosate inhibits?
Shikimate pathway
Which enzyme does Glyphosate inhibit the activity of?
EPSP Synthase
Why is glyphosate not toxic to humans or animals?
Animals do not synthesise their own amino acids, so no pathway to be targeted
How are glyphosate resistance genes found for desirable plants?
Salmonella - AroA mutants grow on glyphosate as they have altered EPSP synthase gene
How are glyphosate resistance genes incorporated into plants?
Place mutant AroA gene in front of a strong promoter (e.g., CAMV 35S) -> every cell expresses the bacterial gene
What is the “better” glyphosate resistance gene that is now used rather than the original E. coli or Salmonella genes?
CP4 from Agrobacterium (retains low Km -> higher affinity for substrate)
What is meant by the “Dual Strategy” for glyphosate resistance?
As well as the CP4 gene, glyphosate oxidase (GOX) isolated from Ochrobactrum antropi allows metabolism of Glyphosate into harmless intermediate (glycine)
What are the main disadvantages of the Transgenic Soybean?
No increase in yield; reliance on one herbicide; glyphosate resistance
What is the first step in cheese production?
Milk precured at warm temperature and ACIDIFIED BY LACTOBACILLI
What is the role of Rennet in cheese production, and what type of recombinant chymosin is generally used today?
Rennet is a protease which removes surface glycopeptides from soluble casein micelles in milk, allowing particle coagulation; recombinant calf mycosin from Aspergillus niger is used
What species of fungus is usually used for surface ripening of cheese?
Penicillium camemberti
What species of fungus is usually used for invasive ripening of cheese?
Penicillium roqueforti
Name the most common top fermenter and what type of drink these are used in
Saccharomycese cervisiae - Ales
Name the most common bottom fermenter and what type of drink these are used in
Saccharomycese carlsbergensis - Lagers
Name the two main types of molecules that give flavour to beers and lagers
Esters (mainly beer) and Fusel Alcohols
Name the species of fungus used to produce most citric acid
Aspergillus niger
Describe the conditions required for production (and purification) of citric acid
Low Manganese and Iron; precipitation or ion exchange; stainless-steel
Name the non-starch polysaccharide in cereals which can increase viscosity in the gut, and the enzyme which is added to alleviate this
Arabinoglucans; ß-glucanase
Name the microbially produced enzyme which is added to animal feed to reduce phosphate supplementation requirement (and the molecule that this enzyme breaks down)
Phytase (phytate)
Name the bacteria which is often found on salads and which pregnant people are most vulnerable to (and the name of the disease it causes)
Listeria monocytogenes (Listeriosis)
Name the bacteria which is often found on chicken and which young/elderly people are vulnerable to
Campylobacter
What are the more serious symptoms of Listeriosis?
Meningitis, Septicaemia, Miscarriage/Stillbirth/Meningitis in Newborn
State the three relevant facts about Listeria monocytogenes and food contamination (sorry bad question but idk how to phrase it)
- Shredding of plants releases saps (which contain sugars for growth of Listeria)
- Listeria can grow at 4C
- if contaminated water/manure added to soil, directly contaminates vegetables and indirectly contaminates milk
Name the potent fungal toxin that killed off many UK turkeys in the 1960s - and the two species that produce it
Aflatoxins -> Aspergillus flavus and Aspergillus parasiticus
Name the three long-term effects of Aflatoxins
Cancer, Liver/Kidney Failure, Brain Damage
Name the species of fungus responsible for Ergot poisoning
Claviceps purpurea
State 4 problems with discharge of untreated sewage into rivers or oceans
- Organic compounds are oxidised by aerobes -> algal blooms, reduction in dissolved O2 -> death of aquatic life
- Solids cause silting of rivers and estuaries
- Toxic substances (e.g. ammonia and heavy metals) -> kill aquatic life and build up in food chain
- Pathogenic bacteria and viruses -> risk to human health
What are the main two elements of sewage that need to be removed?
Organic content and ammonia
How is waste concentration measured? (Name of thing that is measured, and what it represents)
Biological Oxygen Demand (BOD) -> measure of oxidisable organic content by how much oxygen is consumed by bacteria in the dark over a set time
Name the stages of domestic sewage treatment
- Primary Screening
- Primary Settling Tank
- Secondary treatment (percolating filter bed OR activated sludge process)
- Secondary Settling Tank
- Tertiary treatment (sand or reed bed)
What do the microbes in the percolating filter process do?
- Form biofilm on clinker surface
- Those in top 0.5m oxidise organic compounds
- Those blow oxidise NH4 to NO3 (nitrification)
What can happen if the rate of waste spray in the percolating filter process is too high?
Biofilm gets too thick, causing blockages
What is different about activated sludge process compared to percolating filter?
Microbes are not surface attached, and the tank is aerated by air diffusers at the base
What are the advantages and disadvantages of activated sludge process compared to percolating filter?
Up to 10X faster, but running costs higher and more sensitive to composition of incoming water
How are primary and secondary sludge dealt with?
Anaerobic digestion - incubated in sealed stirred tank reactor at 35C, organic matter converted to methane, CO2 and water, sludge cake produced
Name the three types of bacteria involved in Anaerobic Digestion and the stage they are each involved in
FERMENTATIVE BACTERIA (Org matter -> Org acids/esters/alcohols); ACETOGENIC BACTERIA (acids/esters/alcohols -> Acetic Acid + H2 + CO2) METHANOGENS (Acetic Acid + H2 + CO2 -> CH4 +H20)
What is the potential problem with anaerobic digestion that must be monitored?
Methanogens’ doubling time is longer than acetogens + fermentative bacteria; this can lead to increase in H2, causing acetogens to produce longer chain organic compounds (e.g., Butyric acid) which are Not Used By Methanogens
What can sludge cake be used for?
Fertiliser; Incineration to heat anaerobic digester; CH4 + CO2 produced can be used as fuel (bigas)
