Team:Tokyo-NoKoGen

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<h2>Overview</h2>
<h2>Overview</h2>
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<!--<p> Many pesticides use neurotoxins that show toxicity not only to insects but also towards other species, including mammals. We therefore set out to construct an E. coli that can safely eradicate cockroaches but not affect mammals.</p>
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<p> Cockroaches and many other insects store their energy in the form of trehalose and use the enzyme trehalase to convert it to glucose when needed. Our engineered <i>E. coli</i> will overexpress the enzymes OtsA and OtsB to produce trehalose, and glucose-3-dehydrogenase to convert trehalose to 3,3’-diketotrehalose, an inhibitor of trehalase that is not toxic for mammals. Our engineered Exterminator coli will help eradicate roaches from homes without harming humans or pets.</p>-->
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<p> Pesticides are not only harmful to insects, such as cockroaches, but also pose a risk to humans and the environment. A safe pesticide is greatly needed. Our project for iGEM 2014 is “<i>Exterminator coli</i>, an <i>Escherichia coli</i> machine that exterminates insects such as roaches while being completely safe to mammals and the environment.</p><br>
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<p> Pesticides that are harmful to insects, such as cockroaches, put humans and the environment at risk, so safety pesticides are needed. Our project for iGEM 2014 is “Exterminator coli”. We engineered <i>Escherichia coli</i> (<i>E.coli</i>) that produces an inhibitor of a metabolic pathway for insects and exterminates insects such as roaches. In our project, we will produce a pesticide that has no effect on mammals and the environment using <i>E.coli</i>. </p><br>
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<p> Roaches and many other insects store their energy in the form of trehalose, which can be converted to glucose, when needed, by the enzyme trehalase. However, mammals store their energy in the form of glycogen, which does not require trehalase for conversion to glucose. Trehalase inhibitors can therefore be used as a pesticide that can control roaches while being harmless for mammals and the environment.</p><br>
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<p> Roaches and many other insects store their energy in the form of trehalose and use the enzyme trehalase to convert trehalose to glucose when needed. However, mammals store their energy in the form of glycogen, which does not require trehalase for conversion to glucose. Therefore trehalase inhibitor can be used as a pesticide which isn’t harmful to mammals but roaches. </p><br>
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<p> We engineered <i>E. coli</i> that produces the trehalase inhibitor, 3,3´-diketotrehalose (3,3´-dkT), a pesticide that is harmless for the environment and mammals. Our engineered <i>E. coli</i> will overexpress trehalose 6-phosphate synthase (OtsA) and trehalose 6-phosphate phosphatase (OtsB), which convert glucose-6-phosphate and UDP-glucose to trehalose, and glucose-3-dehydrogenase (G3DH), which converts trehalose to 3,3’-dkT. We predict that <i>Exterminator coli</i> will continue to produce 3,3’-dkT even after it is ingested by the insect.</p>
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<p> We engineered <i>E.coli</i> that produces the trehalase inhibitor, 3, 3'-diketotrehalose (3, 3'-dkT), a harmless pesticide to the environment and mammals. Our engineered <i>E.coli</i> will overexpress <i>OtsA</i> and <i>OtsB</i> which convert glucose-6-phosphate and UDP-glucose to trehalose, and glucose-3-dehydrogenase (G3DH) which converts trehalose to 3, 3'-dkT. Therefore, we predict that our <i>E.coli</i> can continue to produce 3, 3'-dkT by using glucose in insects. All we need to do is make roaches eat the <i>E.coli</i> we engineered !</p>
 
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<img src="https://static.igem.org/mediawiki/2014/4/4a/NoKo14_Ecoli.jpg"><br>
<img src="https://static.igem.org/mediawiki/2014/4/4a/NoKo14_Ecoli.jpg"><br>
<img src="https://static.igem.org/mediawiki/2014/4/45/Noko14_Project1.png"><br>
<img src="https://static.igem.org/mediawiki/2014/4/45/Noko14_Project1.png"><br>
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<p>Fig 1. Outline of our project, <i>Exterminator coli</i>.</p><br>
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<p>Fig. 1 Outline of our project, <i>Exterminator coli</i>.</p><br>
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<h2>Why did we focused on pesticide?</h2>
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<h2>Why did we focus on pesticide?</h2>
<p>  Pesticides for insects are used almost everywhere -- not only in agriculture, but also in homes, parks, schools, buildings, forests, and roads. However, it is known that substances included in the pesticide have harmful influence for environment and also to humans. These harmful substances can be diffused in the environment. For example, organophosphates included in the agricultural chemicals affects nervous system, then cause miosis, vomiting, ataxia and muscular twitching. Also, pyrethroid included in spray pesticide does not cause serious poisoning unless it is overdose, but if we absorb surplus pyrethroid, it cause nausea, diarrhea, dizziness and convulsion. Furthermore, boric acid included in poison bait for cockroaches causes dehydration, gastroenteritis and lethargic sleep. There is a fatal case by overdose of boric acid. Therefore, we need pesticide that can exterminate only insects without any effect for environment and humans.</p><br><br>
<p>  Pesticides for insects are used almost everywhere -- not only in agriculture, but also in homes, parks, schools, buildings, forests, and roads. However, it is known that substances included in the pesticide have harmful influence for environment and also to humans. These harmful substances can be diffused in the environment. For example, organophosphates included in the agricultural chemicals affects nervous system, then cause miosis, vomiting, ataxia and muscular twitching. Also, pyrethroid included in spray pesticide does not cause serious poisoning unless it is overdose, but if we absorb surplus pyrethroid, it cause nausea, diarrhea, dizziness and convulsion. Furthermore, boric acid included in poison bait for cockroaches causes dehydration, gastroenteritis and lethargic sleep. There is a fatal case by overdose of boric acid. Therefore, we need pesticide that can exterminate only insects without any effect for environment and humans.</p><br><br>
<h2>Mammal friendly pesticide    -Trehalase inhibitor-</h2>
<h2>Mammal friendly pesticide    -Trehalase inhibitor-</h2>
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<p>  Blood sugar of insects including cockroaches is trehalose which is one of the disaccharide. This means insects store trehalose as source of energy. Insects utilize trehalose in their cell metabolism by reconvering trehalose into glucose. Trehalose is converted to glucose by an enzyme called trehalase (α-glucoside-1-glucohydrolase) which hydrolyses trehalose. Then glucose will be phosphorylated, and used for synthesis of glycogen. Eventually, synthesized glycogen will be catabolized via glycolysis, or pentose phosphate pathway. Although mammals are unable to synthesis trehalose, they can use this sugar if it is present in their diet. In mammals, trehalase is restricted to microvilli membranes of the intestine and the kidney, so there is no indication of regulation of trehalase activity in mammals. Therefore inhibiting trehalase will not effect to mammals.<p><br>
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<p>  Many insects, including cockroaches, store their energy in the form of the disaccharide trehalose in their blood. Insects utilize trehalose in their cell metabolism by reconverting it into glucose by the enzyme trehalase (α-glucoside-1-glucohydrolase), which hydrolyses trehalose (1). The glucose can then be phosphorylated and used for glycogen synthesis or catabolized via glycolysis or the pentose phosphate pathway. Although mammals do not synthesize trehalose, they possess a trehalase in their intestine and kidney, which allows them assimilate trehalose if present in their diet. A trehalose inhibitor will therefore not pose any significant harm to mammals.<p><br>
<img src="https://static.igem.org/mediawiki/2014/d/de/Noko14_Ins1.png" width="80%">
<img src="https://static.igem.org/mediawiki/2014/d/de/Noko14_Ins1.png" width="80%">
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<p>Fig 2.</p>
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<p>Fig. 2 Synthesis of trehalose in insects</p><br><br>
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<p>  In our project, we focused on 3, 3’-diketotrehalose (3,3’-dkT) as trehalase inhibitor. The chemical structure of 3, 3’-dkT resembles trehalose, so the enzyme activity of trehalase will be prevented by competitive inhibition. Since it is reported that it can be synthesized via enzymatic reaction from trehalose, we assumed that we can make <i>E. coli</i> produce 3,3’-dkT and kill insects without harming mammals and environment.  </p><br><br>
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<p>  We discovered from the literature that 3,3’-diketotrehalose (3,3’-dkT) can be used as a trehalase inhibitor (Kojima <i>et al</i>, 2001). Trehalose is converted to 3,3’-dkT by the enzymatic reaction of glucose 3 dehydrogenase (G3DH). The chemical structure of 3,3’-dkT resembles trehalose, so the enzyme activity of trehalase will be prevented by competitive inhibition.  </p><br><br>
<p><b>Reference</b></p>
<p><b>Reference</b></p>
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<p>Becker, A. <i>et al </i>. (1996) The regulation of trehalose metabolism in insects. <i>Experientia </i>, <b>52</b>, 433-439</p>
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<p>(1) Becker, A. <i>et al </i>. (1996) The regulation of trehalose metabolism in insects. <i>Experientia </i>, <b>52</b>, 433-439</p>
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Latest revision as of 01:45, 18 October 2014

Overview

 Pesticides are not only harmful to insects, such as cockroaches, but also pose a risk to humans and the environment. A safe pesticide is greatly needed. Our project for iGEM 2014 is “Exterminator coli”, an Escherichia coli machine that exterminates insects such as roaches while being completely safe to mammals and the environment.


 Roaches and many other insects store their energy in the form of trehalose, which can be converted to glucose, when needed, by the enzyme trehalase. However, mammals store their energy in the form of glycogen, which does not require trehalase for conversion to glucose. Trehalase inhibitors can therefore be used as a pesticide that can control roaches while being harmless for mammals and the environment.


 We engineered E. coli that produces the trehalase inhibitor, 3,3´-diketotrehalose (3,3´-dkT), a pesticide that is harmless for the environment and mammals. Our engineered E. coli will overexpress trehalose 6-phosphate synthase (OtsA) and trehalose 6-phosphate phosphatase (OtsB), which convert glucose-6-phosphate and UDP-glucose to trehalose, and glucose-3-dehydrogenase (G3DH), which converts trehalose to 3,3’-dkT. We predict that Exterminator coli will continue to produce 3,3’-dkT even after it is ingested by the insect.



Fig. 1 Outline of our project, Exterminator coli.



Why did we focus on pesticide?

 Pesticides for insects are used almost everywhere -- not only in agriculture, but also in homes, parks, schools, buildings, forests, and roads. However, it is known that substances included in the pesticide have harmful influence for environment and also to humans. These harmful substances can be diffused in the environment. For example, organophosphates included in the agricultural chemicals affects nervous system, then cause miosis, vomiting, ataxia and muscular twitching. Also, pyrethroid included in spray pesticide does not cause serious poisoning unless it is overdose, but if we absorb surplus pyrethroid, it cause nausea, diarrhea, dizziness and convulsion. Furthermore, boric acid included in poison bait for cockroaches causes dehydration, gastroenteritis and lethargic sleep. There is a fatal case by overdose of boric acid. Therefore, we need pesticide that can exterminate only insects without any effect for environment and humans.



Mammal friendly pesticide -Trehalase inhibitor-

 Many insects, including cockroaches, store their energy in the form of the disaccharide trehalose in their blood. Insects utilize trehalose in their cell metabolism by reconverting it into glucose by the enzyme trehalase (α-glucoside-1-glucohydrolase), which hydrolyses trehalose (1). The glucose can then be phosphorylated and used for glycogen synthesis or catabolized via glycolysis or the pentose phosphate pathway. Although mammals do not synthesize trehalose, they possess a trehalase in their intestine and kidney, which allows them assimilate trehalose if present in their diet. A trehalose inhibitor will therefore not pose any significant harm to mammals.


Fig. 2 Synthesis of trehalose in insects



 We discovered from the literature that 3,3’-diketotrehalose (3,3’-dkT) can be used as a trehalase inhibitor (Kojima et al, 2001). Trehalose is converted to 3,3’-dkT by the enzymatic reaction of glucose 3 dehydrogenase (G3DH). The chemical structure of 3,3’-dkT resembles trehalose, so the enzyme activity of trehalase will be prevented by competitive inhibition.



Reference

(1) Becker, A. et al . (1996) The regulation of trehalose metabolism in insects. Experientia , 52, 433-439