Difference between revisions of "Team:Tokyo-NoKoGen/Project-4"

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<div id="main">
 
<div id="main">
 
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         <div id="box1">
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          <p><m1>Biosafety </m1></p>
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          <p><m4>Experiments of electrode</m4></p>
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        <m1>Light-induced lysis system</m1><br>
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<m3> ExTermite Coli targets to inhibit the metabolic pathway of termite. However, most of insects store their energy in form of trehalose, the same as termite. Therefore, there is one possibility that ExTermite Coli affect other insects. Also, we have to deal with the problem of E. coli spread into the environment. As we use pseudo egg to carry ExTermite Coli to termite’s colony, there is a possibility of distribution and that may cause a genetic pollution.
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        <m3>There is one concern of our ExTermite Coli….Does the ExTermite Coli maintains its function inside termite?<br>
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<br>  To avoid these problems, we add another function to ExTermite Coli….light-induced lysis system.</m3><br>
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　　The main component of ExTermite Coli is G3DH, which catalyzes the reaction to produce toxic material to termite. G3DH is one of an oxidoreductase and it requires substances which can receive electrons to function. Even though the G3DH expressed in E. coli under aerobic condition and can turn over to function correctly, there is no proof of functioning of G3DH in termite’s body.( See Details)<br>
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    However, we think there is a possibility that an electron acceptor for G3DH exists inside termites.<br>
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　　　　　</div>
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    During digestion of cellulose, termite also decomposes lignocellulose, which is contained in wood. One of components of lignocellulose is lignin, which is degradation product of lignocellulose. When lignin is produced, quinone, a possible electron acceptor of G3DH, is also produced.<br>
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　　Based on our hypothesis, we confirmed whether the ExTermite Coli could work in the termite’s gut in the experiments using electrode.<br><m3>
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      <div id="box2">
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      <m1> The reason for constructing “light-induced” lysis system.</m1><br>
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<m3>  One of the most characteristic features of termite is negative phototaxis. When termites such as Coptotermes formosanus exposed to the light, they move very slowly or they stop to move. As for Hodotermopsis sjostedti, they started to run away from the light. Because of termite’s negative phototaxis, ExTermite Coli should be designed to work under dark place.<br>
+
−
  In other words, ExTermite Coli shouldn’t work under light place to avoid damaging other insects outside of house.<br>
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Therefore, to prevent the risk of damaging other insects, we constructed light-induced lysis system.</m3><br>
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        </div>
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        <div id="box3">
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<m1>Mechanism of light-induced lysis system</m1><br>
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<m3>　To construct our biosafety system, we choose two components.<br>
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　First, we choose cpcG2 promoter to induce gene expression by light. CpcG2 promoter originates from cyanobacteria, which can response to green light. In cyanobacteria, cpcG2 promoter function together with other two response regulators, CcaR and CcaS. By introducing this system into E.coli that does not have any photoreceptor, we can induce expression of a specific gene in E.coli.<br>      
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        </div>
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　　　  <div id="box4">
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　<m3>Second, we select the Colicin-E1 as a lysis protein. Colicin-E1 is a membrane protein originating from E.coli. Colicin-E1 forms multimer and binds to receptor on outer membrane to create a pore. It causes membrane disruption and cell lysis.</m3> <br>
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<m3>Using these two components, we constructed light-induced lysis system.</m3><br>
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            </div>
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              <div id="box5">
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<m1>Construction of light-induced lysis gene and evaluation</m1><br>
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<m3>　 First of all, we constructed following Biobricks containing Colicin-E1(BBa_K1765002).<br>
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  We constructed our biobrick from BBa_K822002, which is coding Colicin-E1 and its immune protein. After cloning the genes of Colicin-E1 from this biobrick, we attached T0 terminator and cpcG2 promoter (BBa_K1765002). <br>
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            <center><img src="https://static.igem.org/mediawiki/2015/d/df/Lysis2.jpeg" width="400px"; height="300px";></center>
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　<m2>We could not evaluate the function of this biobrick, but we are going to check the function.</m2><br>
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<m1>　　　　<u><Details：About turn over of G3DH in termite gut ><br></m1></u>
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            </div>
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<m3>      　　G3DH is expected to contain flavin adenine dinucleotide (FAD) as a cofactor because of FAD binding motif in G3DH[1]. FAD is reduced when G3DH oxidizes substrate. Therefore re-oxidation of FAD is required to continuous oxidation of substrate. In E. coli, pyrroloquinoline quinone (PQQ) - dependent D-glucose oxidase, which is an enzyme oxidizes saccharides and has PQQ as a cofactor, donates electron to ubiquinone eight in respiratory chain to reconstitute the PQQ [2]. <br>
 +
　　　　　Therefore, in an aerobic condition, G3DH may donate electron to an electron acceptor in respiratory chain of E. coli and its cofactor may be re-oxidized as PQQ-dependent glucose oxidase. However, as inside of termite gut is anaerobic, the respiratory chain does not work. Therefore, reduced FAD cannot donate electron to electron acceptor.<br>
 +
For that reason, in order that G3DH continues to work in termite gut, extracellular electron acceptor should exist.<br></m3>
 +
   
 +
<m1>    <u> < Details：Electron acceptor in termite gut><br></m1></u>
 +
<m3>　　　　Lignin is contained in woods about 20 ‾ 30 % (w/w) and it is one of the main components of wood the same as cellulose. Lignin can be decomposed by laccase, which exist in the termite’s gut. After decomposed by laccase, quinone is produced in termite’s gut solution. If this quinone can be work as electron acceptor of G3DH, ExTermite Coli can work in termite’s gut.<br></m3>
  
 +
<m1>Why we used electrode to evaluate G3DH activity inside termite’s gut?<br></m1>
 +
<m3> It is said that quinone exists in the termite gut solution. So we think that quinone can work as an electron receptor. <br>
 +
Electron from the reaction of trehalose oxidation was transported from FAD to electron receptor. After an electron receptor received electrons, electron receptor was oxidized and stored on the electrode. And then, after applying potential, an electron receptor was changed from oxidized to reduced form. At that time, electrons are transported to the electrode and we can observe response current.<br>
 +
　 In this experiment, we use screen printed carbon electrode (SPCE). This is one of an electrode used for sensor of medical equipment. Using this type of electrode, we can detect small electrical response to see the activity of enzymes. Also, only little amount of substrate is needed to experiment. It is great advantage for this experiment because we are no able to extract large amount of gut solution from tiny termites.<br></m3>
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　　<img src="https://static.igem.org/mediawiki/2015/b/bb/NK-elec1.jpeg">
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              <div id="box3">
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<m1>　Electrode Experiments<br></m1>
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<m3>　　　First of all, we extracted the gut solution from termite. Then, check the response current with gut solution using SPCE.<br></m3>
  
 +
<m1>            < Details: The method of electrode experiments><br></m1>
 +
<m3>        After obtaining the gut solution, we covered SPCE with 1.5 μL of mixture (1 μL G3DH, 1 μL gut solution and 2 μL glucose) and incubated the electrodes for 60 seconds at room temperature. After incubation, +400 mV vs. Ag/AgCl was applied and we measured the  response current.<br>
 +
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<div id="box4">
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  <img src="https://static.igem.org/mediawiki/2015/a/a4/NK-elec2.jpeg">
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  <div id="box4">
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    <img src="https://static.igem.org/mediawiki/2015/0/00/NK-elec3.jpeg">
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 +
<div id="box5"> 
 +
<m3>Figure (). shows the result of the experiments using electrode. From this result, we could observe apparently high response current when we use termite gut solution in the sample compared to ----. This result showed that G3DH can work in termite’s gut. <br>
 +
All together, it is indicated that ExTermite Coli can produce metabolic pathway inhibitors in termite’s gut.<br></m3>
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Revision as of 17:22, 23 November 2015

　

Experiments of electrode

There is one concern of our ExTermite Coli….Does the ExTermite Coli maintains its function inside termite?
　　The main component of ExTermite Coli is G3DH, which catalyzes the reaction to produce toxic material to termite. G3DH is one of an oxidoreductase and it requires substances which can receive electrons to function. Even though the G3DH expressed in E. coli under aerobic condition and can turn over to function correctly, there is no proof of functioning of G3DH in termite’s body.( See Details)
However, we think there is a possibility that an electron acceptor for G3DH exists inside termites.
During digestion of cellulose, termite also decomposes lignocellulose, which is contained in wood. One of components of lignocellulose is lignin, which is degradation product of lignocellulose. When lignin is produced, quinone, a possible electron acceptor of G3DH, is also produced.
　　Based on our hypothesis, we confirmed whether the ExTermite Coli could work in the termite’s gut in the experiments using electrode.
　　 　　 　　 　　　　
 　　G3DH is expected to contain flavin adenine dinucleotide (FAD) as a cofactor because of FAD binding motif in G3DH[1]. FAD is reduced when G3DH oxidizes substrate. Therefore re-oxidation of FAD is required to continuous oxidation of substrate. In E. coli, pyrroloquinoline quinone (PQQ) - dependent D-glucose oxidase, which is an enzyme oxidizes saccharides and has PQQ as a cofactor, donates electron to ubiquinone eight in respiratory chain to reconstitute the PQQ [2].
　　　　　Therefore, in an aerobic condition, G3DH may donate electron to an electron acceptor in respiratory chain of E. coli and its cofactor may be re-oxidized as PQQ-dependent glucose oxidase. However, as inside of termite gut is anaerobic, the respiratory chain does not work. Therefore, reduced FAD cannot donate electron to electron acceptor.
For that reason, in order that G3DH continues to work in termite gut, extracellular electron acceptor should exist.
 < Details：Electron acceptor in termite gut>
 　　　　Lignin is contained in woods about 20 ‾ 30 % (w/w) and it is one of the main components of wood the same as cellulose. Lignin can be decomposed by laccase, which exist in the termite’s gut. After decomposed by laccase, quinone is produced in termite’s gut solution. If this quinone can be work as electron acceptor of G3DH, ExTermite Coli can work in termite’s gut.
 Why we used electrode to evaluate G3DH activity inside termite’s gut?
 It is said that quinone exists in the termite gut solution. So we think that quinone can work as an electron receptor.
Electron from the reaction of trehalose oxidation was transported from FAD to electron receptor. After an electron receptor received electrons, electron receptor was oxidized and stored on the electrode. And then, after applying potential, an electron receptor was changed from oxidized to reduced form. At that time, electrons are transported to the electrode and we can observe response current.
　 In this experiment, we use screen printed carbon electrode (SPCE). This is one of an electrode used for sensor of medical equipment. Using this type of electrode, we can detect small electrical response to see the activity of enzymes. Also, only little amount of substrate is needed to experiment. It is great advantage for this experiment because we are no able to extract large amount of gut solution from tiny termites.
 　　　　
　　　　
　　 　　
　Electrode Experiments
 　　　First of all, we extracted the gut solution from termite. Then, check the response current with gut solution using SPCE.
 < Details: The method of electrode experiments>
 After obtaining the gut solution, we covered SPCE with 1.5 μL of mixture (1 μL G3DH, 1 μL gut solution and 2 μL glucose) and incubated the electrodes for 60 seconds at room temperature. After incubation, +400 mV vs. Ag/AgCl was applied and we measured the response current.
Figure (). shows the result of the experiments using electrode. From this result, we could observe apparently high response current when we use termite gut solution in the sample compared to ----. This result showed that G3DH can work in termite’s gut.
All together, it is indicated that ExTermite Coli can produce metabolic pathway inhibitors in termite’s gut.