BIO2015: Lecture 8

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  • Created by: LMoney
  • Created on: 13-05-14 11:47
Increasing pressure on insect control methods is due to what?
1) Development of resistance to conventional chemical insecticides 2) Decrease in number of insecticides available for use (EU directive 91/414; Plant Protection Products Regulation 2009) 3) Climate change
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what is heterologous expression?
Heterologous expression refers to the expression of a gene or part of a gene in a host organism, which does not naturally have this gene or gene fragment
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what is a bioassay?
A bioassay involves the use of a live animal (in vivo) or tissue (in vitro) to determine the biological activity of a substance, such as a hormone or drug.
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which organisms has DNA cloning and manipulation for Heterologous Expression be used in, to make insecticides?
E.coli and Yeast systems
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what approaches have been taken to develop novel strategies for producing environmentally-friendly insecticides?
recombinant protein approaches; targeting nutrient transport, and using fusion proteins containing specific insecticidal toxins
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how are natural insecticidal compounds found?
insect bioassays, genome library screening
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what is a fusion protein?
a novel protein produced by combining parts (or all) of pre-existing proteins or peptides
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what is a recombinant expression system?
when combining is done at the DNA level, by engineering coding sequences which encode the fusion protein
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how can combining be done at the protein level?
by using chemical reactions (possibly enzyme-catalysed) to link preformed peptides and polypeptides together
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the concept that domains may correspond to what is thought to have driven accelerated protein evolution by recombining exons from different proteins together?
exons
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why is designing proteins from scratch difficult?
because there is (still) no method for predicting tertiary structure from primary sequence that is not based on sequence similarity
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If we want to produce proteins with properties we desire, what is the best route?
pre-existing proteins
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what can then be used to produce variants, which can be screened for function?
vIn vitro mutagenesis (as part of a directed evolution strategy or by design)
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what is an alternative approach that can result in multi-functional proteins?
simple bolt together approach
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A fusion protein containing complete coding sequences for snowdrop lectin (GNA) and green fluorescent protein (GFP) has the functionality of both proteins? what are these functions?
it will bind to carbohydrates, and fluoresce (and can be used to label cell surfaces by binding to glycoproteins - see later). The fusion protein has novel properties, resulting from the combination of functions.
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what is the approach used to Develop Novel Fusion-Based Biopesticides?
1) Identify Suitable Insecticidal Molecules 2) Identify suitable ‘carrier’ molecules
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what are some examples of insect-derived proteins/peptides that are toxic to insects when injected?
arachnid & parasitoid derived venom peptides; neurohormones
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what is the issue with these toxic proteins?
1) When fed to insects these peptides are prone to proteolytic degradation 2)Equally peptides cannot penetrate the insect cuticle if applied as a spray
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where must these peptides must be able to reach to achieve insecticidal activity?
their site of action (CNS)
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which are the most successful insect killers?
Spiders- venom optimised over a period of 400 million years
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are all spiders harmful to humans?
Very few spiders harmful to humans, but all kill insects- variety of toxins which target CNS
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are spiders to harmful to invertebrates?
some are, spiders have specificity (mammalian vs invertebrates)
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are the toxins small or large peptides?
Small (30-40 a.a) cysteine rich peptides
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what toxin does the Funnel web spider use?
w-Atracotoxin-Hv1a (w=omega)
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what is the action of w-Atracotoxin-Hv1a?
Calcium channel blocker
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what concentration of w-ACTX-Hv1a is need to kill Heliothis virescens (the tobacco budworm)?
< 0.25 nmol/g by injection
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what is the structure of w-ACTX-Hv1a?
37 a.a.; 6 cysteine residues (w-ACTX-1 family)
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Many spider toxins block insect (blank) but have no effect on mammalian (blank)
ion channels
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what is a carrier?
hybrid protein used to transport the insecticidal peptide across the insect gut epithelium to the CNS where it is active.
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what are the advantages of GNA, why does it work as a carrier?
1) Non-toxic to mammals but has insecticidal activity against some insects 2) Resistant to proteolysis in the insect gut
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what is the mechanism of transport?
involves binding of GNA to glycoproteins on the gut surface (endocytosis)
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how could these properties be used to create an insecticide?
By producing a hybrid protein in which a lectin is fused to a toxin, it will be possible to use the lectin as a “carrier”, which will transport the toxin across the insect gut epithelium
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does the lectin (carrier) have to be toxic to the insect?
Not necessarily
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where could these hybrid proteins consequently reach?
the haemolymph, and the site(s) where they exert insecticidal activity
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how was this strategy proved to work?
A construct encoding a hybrid protein in which GNA was fused N-terminally to green fluorescent protein (GFP) was assembled
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which yeast was this recombinant protein expressed in, and which organism was this yeast fed to?
The recombinant protein was expressed in yeast (Pichia pastoris), purified, and fed to larvae of tomato moth (Lacanobia oleracea)
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what was the actual proof?
GFP fluorescence was observed on the surface of cells in the haemolymph of these insects, as a result of transport of GNA-GFP fusion protein across the gut and subsequent binding to haematocytes.
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what are the environmental impacts of GMOs?
1) Gene flow 2) Human Health 3) Biodiversity 4) Soil organisms 5) Non target Arthropods 6) Other non-targets
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where could genes be transferred to?
To other crops / wild relatives
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which non-target arthropods could transgenic/sprayed crops affect?
1) Non-target Lepidoptera 2) Non-target herbivores 3) Pollinators 4) Natural enemies; Parasitoids and Predators
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what are some other non-targets that could be affected?
birds
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Commercialization of Fusion Proteins as Bio-Pesticides is being carried out by which 2 companies?
1) ISAGRO 2) Biopharma Process Systems- funded by Technology Strategy Board (TSB)
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which organisms was The recombinant fusion-based biopesticide Atracotoxin-HV1a fused to GNA (FP5) toxic to?
Toxic to targets pests: Mamestra brassicae and Colorado potato beetle, Non-toxic to beneficial insects: honey bee and the parasitoid Eulophus pennicornis
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what is heterologous expression?

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Heterologous expression refers to the expression of a gene or part of a gene in a host organism, which does not naturally have this gene or gene fragment

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what is a bioassay?

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Card 4

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which organisms has DNA cloning and manipulation for Heterologous Expression be used in, to make insecticides?

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Card 5

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what approaches have been taken to develop novel strategies for producing environmentally-friendly insecticides?

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