Difference between revisions of "Team:NYMU-Taipei/Project-At a Glance"

 
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<h1>At a Glance</h1></div>
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<h1>At a Glance</h1></div>
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<h2 style="margin-top:20px; margin-bottom:10px;">What’s the Problem?</h2><hr /><br />
  
<h1 style="margin-top:30px; margin-bottom:10px;">At a Glance</h1>
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<strong style="font-size:16px;">Chemical insecticides:</strong>
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<p>The purpose of our project is to utilize the a filamentous-fungi-specific CRISPR-Cas9 system 1 in conjunction with an optogenetic system 2 to construct a fast acting kill switch system for genetically engineered fungi, which is becoming ever more popular method for dealing with insect pest around the world. Two plasmids will be constructed, each carrying a module of our two part system. Metarhizium anisopliae ARSEF 549 will be transformed with both plasmids and inoculated on live specimen of Bactrocera dorsalis and Spodoptera litura to test the viability and efficacy of the system. If the system is viable, we think that it could be adapted for other organism for similar purposes.</p>
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<h1 style="margin-top:30px; margin-bottom:10px;">Project Description</h1>
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<p style="font-size:16px;">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>The system will be divided into two modules:</p>
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    <h2>1. Optogenetic activation module:</h2>
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<p>The main working components of this module is the VP-EL222 gene and the Pmcl1 promoter. Both components will be introduced to M. anisopliae on the plasmid pBARGPE1. VP-EL222 is a protein that contains a blue-light inducible LOV domain and a transcriptional activator domain responsible for the activation of the execution module. Pmcl1 is a hemolymph-induced promoter from M. anisopliae that activates when the mycelium enters the hemolymph of its host. Limiting the production of the VP-EL222 protein, which will be under the control of Pmcl1, to only after the fungus has successfully penetrated the hosts’ cuticle. This allows the fungi to proliferate in the darkness of the hemolymph. However, when the fungi reaches the end of its life cycle, it must penetrate the insect’s cuticle from the inside to produce conidia on the surface of the host’s cuticle, which puts the mycelium in contact with sunlight. This is when VP-EL222 proteins will dimerize and activate the CRISPR-Cas9 execution module.</p>
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    <h2>2. CRISPR-Cas9 execution module:</h2>
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    <p>The function of this module is to target several essential genes to reduce the survivability and the competiveness of our GM fungi. These target genes will serve as templates for sgRNA, allowing Cas9 to mutate parts of the genes’ sequences to achieve gene disruption. Currently, we are targeting two genes, MrPHR1 and MrPHR2, which encodes photolysase M. anisopliae. The disruption of these genes will reduce the UV resistance and disrupt the trehalose synthesis of our target fungi 3, thus reducing its survivability. We will also be searching for other genes that are essential to M. ansiopliae’s survival to further reduce our fungi’s survivability.</p>
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    <p>After transforming M. anisopliae, we will be inoculating that carries the kill switch system onto two species of insect, Bactrocera dorsalis and Spodoptera litura, to ensure that the whole system functions as intended.</p>
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<h2 style="margin-top:20px; margin-bottom:10px;">Current Solution</h2><hr /><br />
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<p style="font-size:16px;">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 style="font-size:16px;">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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<h2 style="margin-top:20px; margin-bottom:10px;">Our Biosafety Solution</h2><hr /><br />
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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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<h4 style="margin-top:20px; margin-bottom:10px;">In-Out-Suicide</h4>
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<p style="font-size:16px; white-space:pre-wrap;">  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 by fungal cells within the hemolymph.
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  KillerRed, BBa_K1184000, is a fluorescent protein 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. 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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Latest revision as of 22:09, 17 October 2016

At a Glance

At a Glance



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 by fungal cells within the hemolymph. KillerRed, BBa_K1184000, is a fluorescent protein 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. 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.