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Microbes > Module 5 - Exploiting Microorganisms > Flashcards

Module 5 - Exploiting Microorganisms Flashcards

1
Q

Describe the difference between transgenic and cisgenic

A

Transgenic = genetic material has been transferred between unalike species
Cisgenic = gene has been edited within a single species

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2
Q

Name the genus of bacteria that cause Crown Gall Disease

A

Agrobacterium

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3
Q

What triggers Agrobacterium infection of a plant?

A

When a plant is wounded it releases Acetosyringone - the VirA/G protein products detect this molecule and the Agro moves via chemotaxis towards it

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4
Q

What does virD2 do following activation by VirA/G?

A

It excises the T-DNA from the Ti plasmid and transports it into the plant cell

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5
Q

What do virD4 and virB do in Agro infection?

A

They connect the Agro cell to the plant cell

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6
Q

What does LRR do in the plant defence system?

A

It recognises the flagellum and activates a signalling cascade

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7
Q

Describe the signalling cascade when LRR is activated

A

Phosphorylation of MKK4/5 -> Phosphorylation of VIP1 to VIP1p -> VIP1p enters the nucleus and activates transcription of genes encoding antipathogenic response

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8
Q

What does VirF do in Agro infection?

A

VirF degrades VIP1, preventing antipathogenic genes from being transcribed

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9
Q

What does VirE2 do in Agro infection?

A

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

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10
Q

Once T-DNA is integrated into the plant genome, it promotes the synthesis of which 3 proteins?

A

Auxin, cytokinin and opines

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11
Q

Which 2 genes are removed from the T-DNA region in genetic modification?

A

Genes for hormone and opine biosynthesis

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12
Q

Which 2 genes are inserted into T-DNA in genetic modification?

A

The Gene of Interest, and an antibiotic resistance gene

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13
Q

Why are binary vector systems necessary in genetic engineering using T-DNA?

A

EcoRI (restriction enzyme) has a recognition site of 6 bases (once every 4000), so would cut the Ti plasmid into many inconvenient fragments

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14
Q

What is the first step in transforming plant cells with T-DNA?

A

Co-Cultivation (leaf discs placed on agar plate; solution of Agro carrying binary vector poured on top)

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15
Q

What is the second step in transforming plant cells with T-DNA?

A

Selection using selectable antibiotic marker (e.g., kanamycin)

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16
Q

What is the third step in transforming plant cells with T-DNA?

A

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

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17
Q

What part of the plant is the target for transformation using flower dipping methods?

A

Ovules (in the flowers)

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18
Q

Name the species responsible for the Bt toxin

A

Bacillus thuringiensis

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19
Q

Name the three orders of insects affected by Bt toxin

A

Lepidoptera (Moths/Butterflies), Diptera (Flies), Coleoptera (Beetles)

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20
Q

Why does the Bt toxin only affect insects?

A

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

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21
Q

How useful is dried and sprayed Bt as a direct insecticide?

A

Not very - unstable and only provides protection for a few days

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22
Q

How is Bt toxin made more stable for use on cotton plants?

A

Cloned into another bacterium - Pseudomonas flourescens

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23
Q

How can the Bt toxin be expressed in plants themselves?

A

Place the gene in front of the 35S promoter to transform the plant; if larvae feed on plant they ingest the toxin

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24
Q

Which biosynthetic pathways are generally targeted by herbicides?

A

Vitamin synthesis and amino acid synthesis

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25
Q

State 5 problems with conventional herbicides

A
  1. Hard to distinguish very similar species
  2. Often have poor environmental properties
  3. May need more than one type of herbicide
  4. When used as pre-emergence sprays they can be wasteful
  5. Resistance is a growing problem
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26
Q

What is the name of the pathway that glyphosate inhibits?

A

Shikimate pathway

27
Q

Which enzyme does Glyphosate inhibit the activity of?

A

EPSP Synthase

28
Q

Why is glyphosate not toxic to humans or animals?

A

Animals do not synthesise their own amino acids, so no pathway to be targeted

29
Q

How are glyphosate resistance genes found for desirable plants?

A

Salmonella - AroA mutants grow on glyphosate as they have altered EPSP synthase gene

30
Q

How are glyphosate resistance genes incorporated into plants?

A

Place mutant AroA gene in front of a strong promoter (e.g., CAMV 35S) -> every cell expresses the bacterial gene

31
Q

What is the “better” glyphosate resistance gene that is now used rather than the original E. coli or Salmonella genes?

A

CP4 from Agrobacterium (retains low Km -> higher affinity for substrate)

32
Q

What is meant by the “Dual Strategy” for glyphosate resistance?

A

As well as the CP4 gene, glyphosate oxidase (GOX) isolated from Ochrobactrum antropi allows metabolism of Glyphosate into harmless intermediate (glycine)

33
Q

What are the main disadvantages of the Transgenic Soybean?

A

No increase in yield; reliance on one herbicide; glyphosate resistance

34
Q

What is the first step in cheese production?

A

Milk precured at warm temperature and ACIDIFIED BY LACTOBACILLI

35
Q

What is the role of Rennet in cheese production, and what type of recombinant chymosin is generally used today?

A

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

36
Q

What species of fungus is usually used for surface ripening of cheese?

A

Penicillium camemberti

37
Q

What species of fungus is usually used for invasive ripening of cheese?

A

Penicillium roqueforti

38
Q

Name the most common top fermenter and what type of drink these are used in

A

Saccharomycese cervisiae - Ales

39
Q

Name the most common bottom fermenter and what type of drink these are used in

A

Saccharomycese carlsbergensis - Lagers

40
Q

Name the two main types of molecules that give flavour to beers and lagers

A

Esters (mainly beer) and Fusel Alcohols

41
Q

Name the species of fungus used to produce most citric acid

A

Aspergillus niger

42
Q

Describe the conditions required for production (and purification) of citric acid

A

Low Manganese and Iron; precipitation or ion exchange; stainless-steel

43
Q

Name the non-starch polysaccharide in cereals which can increase viscosity in the gut, and the enzyme which is added to alleviate this

A

Arabinoglucans; ß-glucanase

44
Q

Name the microbially produced enzyme which is added to animal feed to reduce phosphate supplementation requirement (and the molecule that this enzyme breaks down)

A

Phytase (phytate)

45
Q

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)

A

Listeria monocytogenes (Listeriosis)

46
Q

Name the bacteria which is often found on chicken and which young/elderly people are vulnerable to

A

Campylobacter

47
Q

What are the more serious symptoms of Listeriosis?

A

Meningitis, Septicaemia, Miscarriage/Stillbirth/Meningitis in Newborn

48
Q

State the three relevant facts about Listeria monocytogenes and food contamination (sorry bad question but idk how to phrase it)

A
  1. Shredding of plants releases saps (which contain sugars for growth of Listeria)
  2. Listeria can grow at 4C
  3. if contaminated water/manure added to soil, directly contaminates vegetables and indirectly contaminates milk
49
Q

Name the potent fungal toxin that killed off many UK turkeys in the 1960s - and the two species that produce it

A

Aflatoxins -> Aspergillus flavus and Aspergillus parasiticus

50
Q

Name the three long-term effects of Aflatoxins

A

Cancer, Liver/Kidney Failure, Brain Damage

51
Q

Name the species of fungus responsible for Ergot poisoning

A

Claviceps purpurea

52
Q

State 4 problems with discharge of untreated sewage into rivers or oceans

A
  1. Organic compounds are oxidised by aerobes -> algal blooms, reduction in dissolved O2 -> death of aquatic life
  2. Solids cause silting of rivers and estuaries
  3. Toxic substances (e.g. ammonia and heavy metals) -> kill aquatic life and build up in food chain
  4. Pathogenic bacteria and viruses -> risk to human health
53
Q

What are the main two elements of sewage that need to be removed?

A

Organic content and ammonia

54
Q

How is waste concentration measured? (Name of thing that is measured, and what it represents)

A

Biological Oxygen Demand (BOD) -> measure of oxidisable organic content by how much oxygen is consumed by bacteria in the dark over a set time

55
Q

Name the stages of domestic sewage treatment

A
  1. Primary Screening
  2. Primary Settling Tank
  3. Secondary treatment (percolating filter bed OR activated sludge process)
  4. Secondary Settling Tank
  5. Tertiary treatment (sand or reed bed)
56
Q

What do the microbes in the percolating filter process do?

A
  • Form biofilm on clinker surface
  • Those in top 0.5m oxidise organic compounds
  • Those blow oxidise NH4 to NO3 (nitrification)
57
Q

What can happen if the rate of waste spray in the percolating filter process is too high?

A

Biofilm gets too thick, causing blockages

58
Q

What is different about activated sludge process compared to percolating filter?

A

Microbes are not surface attached, and the tank is aerated by air diffusers at the base

59
Q

What are the advantages and disadvantages of activated sludge process compared to percolating filter?

A

Up to 10X faster, but running costs higher and more sensitive to composition of incoming water

60
Q

How are primary and secondary sludge dealt with?

A

Anaerobic digestion - incubated in sealed stirred tank reactor at 35C, organic matter converted to methane, CO2 and water, sludge cake produced

61
Q

Name the three types of bacteria involved in Anaerobic Digestion and the stage they are each involved in

A

FERMENTATIVE BACTERIA (Org matter -> Org acids/esters/alcohols); ACETOGENIC BACTERIA (acids/esters/alcohols -> Acetic Acid + H2 + CO2) METHANOGENS (Acetic Acid + H2 + CO2 -> CH4 +H20)

62
Q

What is the potential problem with anaerobic digestion that must be monitored?

A

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

63
Q

What can sludge cake be used for?

A

Fertiliser; Incineration to heat anaerobic digester; CH4 + CO2 produced can be used as fuel (bigas)

Microbes (7 decks)

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