Bacillus Thuringiensis

Understand limitations of using Bt itself as a bio-insecticide and why expression of the cry gene in different organisms could aid in the use of such an insecticidal protein in insect management?

A limitation of using Bt as a bio-insecticide is that its particular Cry protein is only effective to a certain insect, so other insects not affected by that Cry variant are free to flourish and eat away plants. Another limitation of using Bt itself as a bio-insecticide is that the Cry protein is not protected from UV light exposure, so as the protein is exposed to UV light it is damaged. Expression of the Cry gene in different organisms could aid in the use of such an insecticidal protein in insect management. How? Well, now there is the ability to produce chimeric Cry proteins that increase activity that would not have been selected for in nature. Also, scientists have been able to encapsulate and express the Bt toxin gene in fluorescent cells and in xylem tissues which solved the problem of UV light exposure.

What practices and/or genetic processes can interfere with the success of the high dos/ refuge strategy for managing insect resistance to Bt cry toxin?

A practice that can interfere with the success of the high-dose and refuge strategy for managing insect resistance to Bt cry toxin is the act of not complying to maintain at least 20% of total crops to being a refuge area. When farmers do not comply the strategy falls apart and resistant insects emerge successfully. A genetic process that interferes with the success of this strategy is the emergence of non-recessive mutations of Bt toxin resistance.

Understand the processes by which a transgenic plant is made, and how those harboring the Bt cry gene are identified?

Creating a transgenic plant using a Ti plasmid is done by replacing pre-existing oncogenic (causing development of tumors) sequences with desired genes, and placing marker genes so to kill cells that were unsuccessful in acquiring and expressing the desired genes. Creating a transgenic plant through exposure is done by the following steps: 1. cutting the leaf and exposing it to the bacteria carrying the gene of interest and an antibiotic gene 2. once have allowed the genes to enter the plant cell , expose the plant cells to an antibiotic so cells bearing the gene of interest only survive. 3. allow the callus produced in step 2 to sprout into the plant- given that most plant cell are totipotent. Creating a transgenic plant by using a "gene gun," is a process that allows regeneration in a plant by directly introducing plasmid DNA that are carried by tiny porous gold spheres.

What genetic processes complicate successful expression of genes in transgenic organisms when they originate in another kingdom of life?

Genetic processes that complicate successful expression of genes in transgenic organisms when they originate in another kingdom of life are intron splicing that inactivates expression of the gene, and improper codon usage due to the fact that codons differ from Bt to plants.

Compare and contrast the different strategies in which the Bt cry gene was introduced into other microorganisms in an effort to facilitate insect management without resorting to the use of transgenic plants- and what biological/ecological issues limited such approaches?

In algae, expression of the cry protein here was successful in being potent to mosquitoes and in being potent under UV light conditions- but there is uncertainty whether genetically modifying algae producing these crystal proteins will cause an environmental problem. In bacteria thriving on roots, the idea was to perform pest management on root feeding insects. The process was successful but they soon found that there was a threshold of the percentage of insect mortality and yield loss due to other bacteria acquiring the gene and eating away the roots. In bacteria thriving in xylem tissue, has allowed for the protein to thrive in conditions not exposed to UV light but soon held loss due to bacteria arose.

In what ways is agriculture and the management of insect pests one of the most prominent environmental hazards associated with human existence?

Physically, agriculture is an environmental hazard because it has lead to numerous species being endangered as more and more of the land is being turned into agriculture production; currently, 40% of the land surface has been given over to agriculture. On top of that, push to increase operational yield has introduced pest control. This energy in pest control has led to natural selection and introduction of non-natural occurring genes which threatens biodiversity.

Explain how, in principle, the high dose/ refuge strategy should delay the emergence of resistance to Bt cry protein in insects feeding on transgenic insecticidal plants?

Since the genotype of resistance in insects is recessive, then the high-dose and refuge strategy has delayed the emergence of resistance to Bt Cry protein in insects feeding on transgenic insecticidal plants. High dosage ensures that all those that can be killed are, and the refuge ensures that the rate at which resistant insects emerge slows down since there is still available mating with non-resistant insects.

Whats practices and genetic/biochemical mechanisms lead to resistance of insects to the Bt cry protein?

Strategies that lead to resistance in insects were temporal rotation of toxins, and spatial mixture of toxins in the genome. These factors allowed for insects to grow resistance as the number of crops expressing the Bt toxin gene increased.

Understand the biochemical processes by which the Bt cry protein kills insects and why this leads to its selective killing of insects but not other organisms, and thus contributes to its biosafety for humans?

The Bt cry protein must be processed under alkaline conditions to become activated, and the only organisms capable of doing so are insects with their alkaline guts. Once activated the protein binds to the cadherin receptor, inserts itself into the membrane, and with an adjacent protein forms a pore that allows ions to move about freely. Since ions are able to move freely through the gut the insect becomes sick and then dies. Humans do not have an alkaline gut that would activate the protein, since the protein would enter the acidic stomach and become denatured. Therefore, Bt toxin is safe for humans.

Understand the genetic basis for cry protein production in Bt and why this facilitated cloning of the gene responsible for this useful trait and also enables improvement upon mother nature for useful forms for this toxic protein to be produced?

The genetic basis for cry protein production in Bt is plasmids. The Cry protein is harbored on plasmids which has enabled easy cloning and has facilitated production of novel combinations of toxins in a bacterial strain. Because plasmids undergo processes like conjugation, scientists have been able to manipulate conjugation into producing novel combinations of toxins (also known as chimeric Cry proteins) that carry various strains of the protein. Now scientists have the ability to improve upon mother nature for useful forms of the protein, which advances the productivity of the protein upon various insects.