Chapter 16 Flashcards
What are the advantages of biological insecticides over chemical insecticides?
Advantages: highly specific for target insect species, biodegradable, slow to select for resistance
Disadvantages: low potency, high cost of production
Why is the B. thuingiensis toxin not toxic to humans?
Because, the toxin is only active at a pH of 7 whereas human gut pH is at 4 which will inactivate the toxin
Outline a strategy that you would use to isolate an insecticidal protoxin gene from B. thuringiensis israelensis. How would you use this gene in a practical way?
Transfer clone bank of Bt israelnesis into E. coli and plate onto nitrocellulose membrane and lyse cells with organic solvents.
Treat membrane with bovine serum albumin to prevent nonspecific binding and wash with rabbit antiserum against Cry. Wash membrane to remove unbound antibodies and treat with Protein A of S. aureus.
Visualize toxin with autoradiography. The CytA toxin can be used in tandem with other Cry proteins for decrease in insect resistance and increase in toxicity.
How would you determine whether a particular insecticidal protoxin gene is present on a plasmid or part of the chromosome of a B. thuringiensis strain?
Bacterial DNA preparation on CsCl centrifugation tube which will separate chromosomal from plasmid DNA. Isolate the plasmid DNA and sucrose gradient centrifuge it to separate plasmid by size. Discard the small plasmid, digest medium and large plasmids with RE and clone onto pBR322. Transform into E. coli and screen for cry proteins.
How would you use genetic engineering to improve the usefulness of a particular B. thuringiensis protoxin?
To improve the usefulness of a particular Bt protoxin, place the toxin gene under the control of tet promoter (constitutive) and insert the plasmid into a sporulation deficient strain of Bt so that the toxin can be produced even in the absence of sporulation.
If cry1C-cry1Ab fusion gene encoding an insecticidal protoxin consists of approximately 2,200bp of DNA from cry1C gene and 1,300bp of DNA from cry1Ab gene, what is the advantage of synthesizing this fusion protoxin compared with cry1C?
The advantage of this fusion protoxin is that it is much more stable and the toxin is more resistant to UV because it is not secreted out of the cell
How can insect gut enzymes be limited to the processing the B. thuringiensis insecticidal protoxin to the active toxin without degrading the toxin?
Change the amino acid residue Leu 144 to Asp, Ala, Gly or Val for higher yield of active to prevent further proteolytic cleavage by enzymes in gut that would inactivate the toxin.
How would you engineer a Cry protein to lessen or avoid the development of insect resistance to this toxin?
Toxin can be modified to bind to more than one gut epithelial receptor protein which would decrease resistance since insect would have to mutate multiple genes to becomes resistant.
Shuffle different binding specificities and toxins to create novel toxins that can be used in tandem.
Why is the bacterium A. excentricus an attractive host organism for the expression of B. thuringiensis insecticidal toxin genes?
Does not sink, inexpensive, low level of proteases, less sensitive to high levels of UV
How can the species range of an insecticidal B. thuringiensis strain be extended?
Transferring toxin gene from one strain of Bt to a different strain of Bt that targets a different insect
Fusion portions of two different species specific toxin gene to form a hybrid toxin that is dual acting
Modify the portion of the insecticidal toxin responsible for binding to insect gut epithelial cells.
What is a truncated B. thuringiensis insecticidal protoxin?
Removing the C-terminal half of the protoxin (because it is non-toxic) to save resources.
To increase stability, add genes known to enhance synthesis (chaperone proteins) and crystallization of protoxin.
Why is it unlikely that insects will ever develop a resistance to the israelensis strains?
It synthesizes 3 different Cry proteins and produces a CytA endotoxin different from Cry proteins. The CytA toxin attacks lipid component of membrane and can act as a receptor for other Cry proteins.
What are cadherins and how can they be used to enhance the toxicity of a particular Cry protein?
Cry toxin bind to cadherins or receptors on the microvilli of insect gut epithelial cells. Cadherins (12 domains and membrane-proximal extracellular domain) can be added to Cry proteins to increase binding, the cadherin binds to the microvilli and attracts the Cry molecule.
