Difference between revisions of "Team:NYMU-Taipei/Project-Experiment"

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<h1 style="font-size:72px; white-space:pre; color:white;">   TITLE</h1><hr />
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<h1>Experimentd</h1></div>
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<p style="white-space:pre; color:white;">                    Besides the fungal killing switch and the functional prototype that help reduce concerns over GMO</p>
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<h1>Experiment</h1></div>
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<h1>Background</h1><hr /><br />
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<h2 style="margin-top:20px; margin-bottom:10px;">What’s the Problem?</h2><hr /><br />
  
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<p style="font-size:16px;">Our project is comprised of four parts,  the introduction, part design, prototype, and human practice. Let me introduce some background information regarding our project. The Oriental fruit fly is a serious agricultural pest. It can consume more than 150 types of vegetables and fruits and primarily affects tropical areas. According to research, in 2004, Asia produced 178 million tons of tropical fruits, amounting to 66% of the global production worth US$2.5 billion. Because Asia is mainly a tropical environment, these fruit flies can decimate the agricultural industry. They can cause 90% to 100% yield loss depending on the fruit fly population, locality, variety and season. In other words, the oriental fruit fly poses a great threat to our global food supply as it can destroy most of the global produce.</p>
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<b>Chemical insecticides:</b>
  
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<p>Although chemical insecticides have improved the lives of countless human beings by controlling the population of both agricultural and urban pests, since the second half of last century, the numbers of insecticide resistant pests have been rising at an alarming rate [1]. Some would argue that as researchers and chemical pesticide companies develop new insecticides, the resistance couldn’t possibly catch up. In reality, the amount of insecticides applied is actually proportional to the increase in resistance in the target pest population [2, 3]. This means if no action was taken to change the status quo, the ongoing population control of insect pest will become even more of a struggle.</p>
  
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        <p style="font-size:16px;">Oriental fruit flies are wreaking havoc worldwide. It accounted for an annual loss of $176 million in California and billions worth of damage in Taiwan. Furthermore, it devastated fruit production in Africa, causing more than 80% of crop damage. As a result, various countries, out of fear, impose trade restrictions and refused imports of produce from Africa, leading to significant economic losses for Africa.</p>
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        <p style="font-size:16px;">The ravages of oriental fruit flies are also not limited to tropical areas. This is a global issue. They have plagued approximately 60 countries worldwide and the issue is worsened by international transportation, which can spread the oriental fruit fly eggs. This is why the issue of oriental fruit flies needs to be solved and our project aims to solve this problem.</p>
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        <p style="font-size:16px;">Here are a few traditional solutions to the oriental fruit fly, but each of them are far from perfect.</p>
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        <p style="font-size:16px;">#1: One of the most effective approach is contraception or male annihilation, which uses high-energy radiation to sterilize the male and in turn prevent reproduction. This is ineffective, however, as wild female flies can differentiate between sterilized males and non-sterilized males.</p>
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        <p style="font-size:16px;">#2: Another way is baiting, which uses pheromones like methyl eugenol to attract male oriental fruit flies into traps that kill them. This is still ineffective however, as the female fruit flies, being the one capable of reproducing, are the real problem but are unaffected by methyl eugenol. There are other more ways like bagging, spraying pesticides and early harvesting but none of these are perfect solutions.</p>
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        <p style="font-size:16px;">Our project aims to solve the shortcomings of these previous solutions. Our fungi is a biological agent that takes biosafety and environmental conservation into consideration.</p>
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<h2 style="margin-top:20px; margin-bottom:10px;">Current Solution</h2><hr /><br />
  
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<p>Biopesticides, including insecticidal plant extract, bacteria, and fungi, are some of the more popular alternatives to chemical pesticides. Currently, the biopesticides that are the most widespread, in terms of usage, are the entomopathogenic-fungi insecticides [4]. Certain species of entomopathogenic fungi are capable of targeting a small range of hosts, making them the ideal solution to many regional insect pests. However, these biological control agents come with highly variable outcomes due to the variation in environmental (e.g. temperature and humidity) and host (e.g. nutrition and immune response) conditions [5].</p>
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<p>Many researchers have tried to improve these fungal insecticides through biochemical or genetic means [6, 7]. Though they might have achieved in increasing the potency of or decreased the environment’s effects on a fungus species, they did not take a step further to consider the evaluation the fungal insecticide must follow before commercialization, which includes the assessment of its toxicity towards humans and animals, dispersal, horizontal gene transfer, and effects on the resident microflora [5].</p><br />
  
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        <p style="font-size:16px;">What is Metarhizium anisopliae?</p>
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<h2 style="margin-top:20px; margin-bottom:10px;">Our Biosafety Solution</h2><hr /><br />
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        <p style="font-size:16px;">Metarhizium Anisopliae is an entomogenous fungi, Or a fungi that can act as a parasite and seriously harm them.It can infect over 300 hosts but different strands have highly specific hosts. Because various strains of Metarhizium Anisopliae already exist in soil, it doesn’t alter the environment, making it eco-friendly.</p>
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  The lack of biosafety development for genetically engineered fungal insecticides hinders its commercialization and public acceptance. To address this problem, we, 2016 NYMU_Taipei, are designing light-induced kill switch aimed to reduce the dispersal and horizontal gene transfer of genetically engineered fungal insecticides. Using an entomopathogenic fungus that is applied as an insecticide around the world, Metarhizium anisopliae, as our chassis, we constructed a genetically modified M. anisopliae with wildtype lethality and the additional ability to self-terminate after killing its host.<p/>
  
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<h5>In-Out-Suicide<h5><br/>
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  Through synchronizing the different stages of the kill switch activation with that of the M. ansiopliae’s infection cycle, our genetically modified M. anisopliae could infect and kill pest insects before self-termination. This allows for researchers to create highly lethal and environment tolerant fungal pesticides with reduced residual specimen in the area of application.
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  M. anisopliae’s infection cycle starts with conidia adhesion to the cuticle of the host. The fungus will then attempt to penetrate the cuticle by secreting various proteases, chitinases, and lipases. When the mycelium reaches the hemolymph, it will start to produce yeast-like blastospores, which will change gene expression alongside with any fungal cells that is in contact with the hemolymph. This is also when the production phase of our kill switch begins. Utilizing a hemolymph induced promoter from M. anisopliae, Pmcl 1, with fast activation and high production rate, original transcripts of the gene controlled by this promoter could be detected within 20 minutes of cellular contact with insect hemolymph and amounts for 5.6% of all ESTs [8], high amounts of KillerRed protein will be produced fungal cells within the hemolymph.
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  KillerRed, BBa_K1184000, are fluorescent proteins that can be activated by yellow-orange light (540-585 nm) to produce highly reactive oxygen species (O2․-). During M. anisopliae’s stay within the hemolymph and other interior organs of the insect, KillerRed molecules will remain inert due to the lack of yellow light. However, when the fungus depletes the nutrients in its host, M. anisopliae will emerge for conidiation. This put fungal cells in direct contact with sunlight, allowing KillerRed to create O2․- and disrupt the metabolism and eventually killing the fungi.
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  In conclusion, our In-Out-Suicide system allows for more lethal genetically modified fungal pesticides to be developed because it provides them with an post-deployment clean up, lowering their threat to the surrounding environment.
  
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        <p style="font-size:16px;">M. Anisopliae however, is vulnerable to environmental stress like low humidity, temperature, UV exposure… Etc. Thus, scientists are currently genetically engineering M. Anisopliae to solve these shortcomings. But, because of certain laws and policies that bar GMO products from entering the market, biopesticides using M. Anisopliae is still uncommon.</p>
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        <p style="font-size:16px;">Traditionally, biosafety is achieved by selecting strains with low UV tolerance or low heat tolerance to ensure that it dies under uncontrolled environments. This, however, compromises on its virulence and applicability. Thus, our project aims to mitigate these GMO concerns without compromising its virulence and vitality.</p>
 
  
 
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Revision as of 11:52, 16 October 2016

Experimentd

Experiment


What’s the Problem?


Chemical insecticides:

Although chemical insecticides have improved the lives of countless human beings by controlling the population of both agricultural and urban pests, since the second half of last century, the numbers of insecticide resistant pests have been rising at an alarming rate [1]. Some would argue that as researchers and chemical pesticide companies develop new insecticides, the resistance couldn’t possibly catch up. In reality, the amount of insecticides applied is actually proportional to the increase in resistance in the target pest population [2, 3]. This means if no action was taken to change the status quo, the ongoing population control of insect pest will become even more of a struggle.

Current Solution


Biopesticides, including insecticidal plant extract, bacteria, and fungi, are some of the more popular alternatives to chemical pesticides. Currently, the biopesticides that are the most widespread, in terms of usage, are the entomopathogenic-fungi insecticides [4]. Certain species of entomopathogenic fungi are capable of targeting a small range of hosts, making them the ideal solution to many regional insect pests. However, these biological control agents come with highly variable outcomes due to the variation in environmental (e.g. temperature and humidity) and host (e.g. nutrition and immune response) conditions [5].

Many researchers have tried to improve these fungal insecticides through biochemical or genetic means [6, 7]. Though they might have achieved in increasing the potency of or decreased the environment’s effects on a fungus species, they did not take a step further to consider the evaluation the fungal insecticide must follow before commercialization, which includes the assessment of its toxicity towards humans and animals, dispersal, horizontal gene transfer, and effects on the resident microflora [5].


Our Biosafety Solution


The lack of biosafety development for genetically engineered fungal insecticides hinders its commercialization and public acceptance. To address this problem, we, 2016 NYMU_Taipei, are designing light-induced kill switch aimed to reduce the dispersal and horizontal gene transfer of genetically engineered fungal insecticides. Using an entomopathogenic fungus that is applied as an insecticide around the world, Metarhizium anisopliae, as our chassis, we constructed a genetically modified M. anisopliae with wildtype lethality and the additional ability to self-terminate after killing its host.

In-Out-Suicide

Through synchronizing the different stages of the kill switch activation with that of the M. ansiopliae’s infection cycle, our genetically modified M. anisopliae could infect and kill pest insects before self-termination. This allows for researchers to create highly lethal and environment tolerant fungal pesticides with reduced residual specimen in the area of application. M. anisopliae’s infection cycle starts with conidia adhesion to the cuticle of the host. The fungus will then attempt to penetrate the cuticle by secreting various proteases, chitinases, and lipases. When the mycelium reaches the hemolymph, it will start to produce yeast-like blastospores, which will change gene expression alongside with any fungal cells that is in contact with the hemolymph. This is also when the production phase of our kill switch begins. Utilizing a hemolymph induced promoter from M. anisopliae, Pmcl 1, with fast activation and high production rate, original transcripts of the gene controlled by this promoter could be detected within 20 minutes of cellular contact with insect hemolymph and amounts for 5.6% of all ESTs [8], high amounts of KillerRed protein will be produced fungal cells within the hemolymph. KillerRed, BBa_K1184000, are fluorescent proteins that can be activated by yellow-orange light (540-585 nm) to produce highly reactive oxygen species (O2․-). During M. anisopliae’s stay within the hemolymph and other interior organs of the insect, KillerRed molecules will remain inert due to the lack of yellow light. However, when the fungus depletes the nutrients in its host, M. anisopliae will emerge for conidiation. This put fungal cells in direct contact with sunlight, allowing KillerRed to create O2․- and disrupt the metabolism and eventually killing the fungi. In conclusion, our In-Out-Suicide system allows for more lethal genetically modified fungal pesticides to be developed because it provides them with an post-deployment clean up, lowering their threat to the surrounding environment.