Difference between revisions of "Team:Tokyo-NoKoGen/Project-1"

 
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<div id="main">
 
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          <p><m1>Pseudo Egg </m1></p>
+
          <p><m1>&nbsp;&nbsp;Pseudo Egg         </m1></p>
−
          <p><m2>Project</m2></p>
+
 
 
<m3>　　ExTermite Coli is a new system to exterminate termites.</m3>
 
<m3>　　ExTermite Coli is a new system to exterminate termites.</m3>
 
<m3> It is composed of two parts, pseudo egg and engineered E. coli. </m3>
 
<m3> It is composed of two parts, pseudo egg and engineered E. coli. </m3>
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 +
<br>
 +
<br>
 +
<br>
 +
<m2>Pseudo Egg</m2><br>
 +
<m3> Termites live in dark and humid areas, often in the recess of a house, where we cannot reach. Finding termite colony is therefore very difficult.<br>
 +
We have to distribute our ExTermite Coli to kill termites more effectively.. One of our sponsor company,<b> “ASANTE“</b>, who investigates effective way to exterminate harmful insects, uses dogs to detect termites. It is an innovative way, but distribution of pesticides after finding their colony is still difficult. To produce a new system to exterminate termites, we have to deal with this problem. Our answer is…Pseudo egg.</m3><br>
 +
<br>
 +
<m2>What is Pseudo egg?</m2><br>
 +
<m3> Termite is a social insect that forms colonies composed of a queen and workers. One of the main roles of the queen is the production of eggs, but the queen does not take care of her eggs or children by herself. Instead, the worker termites carry eggs, which drop out around the colony, and take care of them by licking them to moisturize and disinfect them. <br>
 +
  Worker termites recognize their egg by its size and an egg pheromone. If they find something spherical coated by egg pheromone, they recognize it as “their egg” and they carry it []. If we make something like that, it will be recognized as their egg, and they will carry it to their colony. Therefore, by making pseudo egg containing our engineered <i>E. coli</i>, our <i>E. coli</i> will be carried into termite’s colony and distributed considering their remarkable characteristic of egg-carrying and egg-licking.
 +
<br>
  
−
<A href="JavaScript:hiraku('たたむ部分の名称');"><Form><Input type="button" Style=" color:#336600; background-color:#ccffcc; border-width:2; height: 30px; font-size: 18px; font-family:fantasy;" value="<Details:詳細のタイトルとか>"></form></A><br>
+
<A href="JavaScript:hiraku('TRE');"><Form><Input type="button" Style=" color:#336600; background-color:#ccffcc; border-width:2; height: 30px; font-size: 18px; font-family:fantasy;" value="<Details: Termites recognize their egg by the following features>"></form></A>
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<DIV CLASS="tuzukiwaku" ID="たたむ部分の名称">
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<DIV CLASS="tuzukiwaku" ID="TRE">
 +
<dl>
 +
<dt><b>Termites recognize their egg by the following features</b></dt>
 +
<dd>
 +
<ul type="disc">
 +
<li>Releasing β-glucosidase and lysozyme as egg hormone
 +
<li>Spherical
 +
<li>0.4-0.5 mm diameter
 +
</ul>
 +
</dd>
 +
</dl>
 +
<A href="JavaScript:tatamu('TRE');">close</A>
 +
</DIV>
 +
</m3>
 +
<br>
 +
<m3> Worker termites also lick the eggs after carrying them into their colony. Actually, the termite’s saliva and egg pheromone contains the same substances. Licking helps to maintain the egg’s proper moisture levels and keep viruses away. Additionally, worker termites have a habit to grooming each other to exchange substances in their gut.  These instincts are useful to deliver ExTermite Coli into the termite’s body.</m3>
 +
<br>
 +
<br>
 +
<m3> We focused on these termite’s instincts, and we made pseudo egg.<br>
 +
    To investigate if we can make engineered E. coli distributed using pseudo egg, we did some experiments.<br>
 +
<br>
 +
  <center><m2><Font Color="#ff0000"> ＊Caution!!＊: Following pages may contain some pictures of termites.<br>
 +
            If you dislike insects, discretion is advised when viewing the “Details” section.</Font></m2></center>
 +
<br>
 +
<A href="JavaScript:hiraku('maintained');"><Form><Input type="button" Style=" color:#336600; background-color:#ccffcc; border-width:2; height: 30px; font-size: 18px; font-family:fantasy;" value="<Details: maintained termites>"></form></A>
 +
<DIV CLASS="tuzukiwaku" ID="maintained">
 +
<dl>
 +
<dt><b>maintained termites</b></dt>
 +
<dd>
 +
<center>We have maintained the following kinds of termites for our experiments.</center><br>
 +
<br>
 +
<b>・Formosan subterranean termite <i>( Coptotermes formosanus )</i> (イエシロアリ)</b><br>
 +
<img src="https://static.igem.org/mediawiki/2015/9/94/Ieshiro.jpg" style=position:"relative"; top:"10px"; right:"10px"; width="360px"; height="335px";><br>
 +
  This termite cause the greatest problem to buildings. They can eat wood, paper, and can also damage concrete buildings. Their reproductive power is so strong that it can form huge colonies consisting of one or two million termites.<br>
 +
<br>
 +
<br>
 +
<b>・Japanese subterranean termite <i>(Reticulitermes speratus)</i> (ヤマトシロアリ)</b><br>
 +
<img src="https://static.igem.org/mediawiki/2015/6/6b/Yamato.jpg" style=position:"relative"; top:"10px"; right:"10px"; width="360px"; height="327px";><br>
 +
Japanese subterranean termite has the largest distribution in Japan.This species of termite does not have strong reproductive power relative to Formosan subterranean termite, but it is resistant to cold.<br>
 +
<br>
 +
<br>
 +
<b>・Japanese damp-wood termite<i> (Hodotermopsis sjostedti)</i>(オオシロアリ)</b><br>
 +
<img src="https://static.igem.org/mediawiki/2015/1/1b/Ooshiro.jpg" style=position:"relative"; top:"10px"; right:"10px"; width="501px"; height="332px";><br>
 +
  The most characteristic feature of Japanese damp-wood termite is its small size. While the above two species are 3-5mm long, Japanese damp-wood termite is 1-1.5 mm long. This species of termite is distributed in the south area of Japan.<br>
 +
<br>   <br>
 +
</dd>
 +
</dl>
 +
<A href="JavaScript:tatamu('maintained');">close</A>
 +
</DIV>
 +
</m3>
 +
<br>
 +
<center><h2>We would like to express our gratitude for ASANTE for provided these termites.</h2></center>
 +
<br>
 +
<br>
 +
<m3> Using these three kinds of termites, we did experiments to confirm whether our pseudo eggs were carried by termites.<br>
 +
First, we made pseudo eggs consisting of sodium alginate, CaCl2, and LB medium. Dripping solution of sodium alginate into CaCl<sub>2</sub> solution makes tiny beads coated by thin film of calcium alginate. LB medium added to the mixture remains inside these tiny beads.</m3> <br>
 +
<br>
 +
<center><img src="https://static.igem.org/mediawiki/2015/7/7b/Egg2.jpg" style=position:"relative"; top:"100px"; right:"10px"; width="432px"; height="225px";></center><br>
 +
<br>
 +
<A href="JavaScript:hiraku('making');"><Form><Input type="button" Style=" color:#336600; background-color:#ccffcc; border-width:2; height: 30px; font-size: 18px; font-family:fantasy;" value="<Details: Making Pseudo Egg>"></form></A>
 +
<DIV CLASS="tuzukiwaku" ID="making">
 
<dl>
 
<dl>
−
<dt><b>詳細</b></dt>
+
<dt><b>Making Pseudo Egg</b></dt>
 
<dd>
 
<dd>
−
  ほんとうに折りたためるのか？<br>
+
  Mixture of sodium alginate (10% w/w) and LB medium was dripped into a solution of CaCl<sub>2</sub> by cylinder (the internal diameter：0.5mm). <br>
−
  <br>
+
After making tiny beads, they were sorted by their size and 0.4 ~ 0.5 mm diameter beads were selected for experiments.<br>
 +
                Selected beads were then coated with egg pheromone (1 µg/mL β-glucosidase and 1 µg/mL lysozyme). <br>
 +
<center><img src="https://static.igem.org/mediawiki/2015/6/6d/Egg1.jpeg" style=position:"relative"; top:"100px"; right:"10px"; width="390px"; height="297px";></center>
 +
<br>
 
</dd>
 
</dd>
 
</dl>
 
</dl>
−
<A href="JavaScript:tatamu('たたむ部分の名称');">close</A>
+
<A href="JavaScript:tatamu('making');">close</A>
 +
</DIV>
 +
<br>
 +
<m3> Pseudo eggs were put on the petri dish with wet Kimtowel and nine Formosan subterranean termites released into the dish. This dish was observed for one week, and then we counted the number of the pseudo egg to check whether the pseudo eggs were moved. Fig(3) shows the result of this experiment. As a result, we find out that the number of pseudo eggs coated by pheromone was decreased compared to that of control pseudo eggs.</m3>
 +
<br>
 +
<center><img src="https://static.igem.org/mediawiki/2015/9/97/Egg3.jpeg" style=position:"relative"; top:"100px"; right:"10px"; width="450px"; height="390px";></center><br>
 +
<br>
 +
<br>
 +
<m3>　Next, we checked whether the <i>E. coli</i> remains attached on pseudo eggs.To carry ExTermite Coli to termites by using pseudo egg, E. coli should remain attached on the surface of pseudo eggs. Also, <i>E. coli</i> must remain viable for a long time, until it reaches a termite’s gut. We checked these two concerns by the following experiments.<br>
 +
　Figure (4) shows the scheme of experiment using RFP-expressing <i>E. coli</i>. We made an E. coli that expresses red fluorescence protein (RFP) and mixed it with pseudo eggs. Then, we put the pseudo eggs on a LB agarose plate to check whether inside <i>E. coli</i> can come out from the pseudo eggs. After 24 h incubation at 37℃, we moved the pseudo eggs to the next square place. If the RFP-expressing <i>E. coli</i> can come out from the pseudo egg, red colony will be formed. Also, if the RFP-expressing <i>E. coli</i> can live on the pseudo egg, red colony will be formed on the second square place.<br>
 +
<center><img src="https://static.igem.org/mediawiki/2015/0/00/Egg4.jpeg" style=position:"relative"; top:"100px"; right:"10px"; width="450px"; height="365px";></center><br>
 +
　From the Figure (4), we could find that the E. coli could come out from the pseudo egg. It is remarkable that <i>E. coli</i> can come out from inside of the pseudo egg. <br>
 +
<br>
 +
<br> 
 +
　Finally, to check whether <i>E. coli</i> can be alive on the surface of the pseudo egg, we also made pseudo eggs which were coated by RFP-expressing <i>E. coli</i> and set on a LB agarose plate in the same way as above. After 24 h incubation at 37℃, we moved the pseudo eggs to the next square place. If the RFP-expressing <i>E. coli</i> can live on the pseudo egg, red colony will be formed on the second square place.<br>
 +
<br>　　
 +
<center><img src="https://static.igem.org/mediawiki/2015/8/83/Egg5.jpeg" style=position:"relative"; top:"100px"; right:"10px"; width="572px"; height="387px";></center><br>
 +
<br>
 +
　As a result, we found that RFP-expressing <i>E. coli</i> coated outside of the pseudo egg can live for two days on the surface of the pseudo egg. That means by this method of making, pseudo egg can carry <i>E. coli</i> for some days to distribute E. coli between termites. We are now trying to lengthen the period E. coli can survive by examining the components of pseudo egg.</m3>
  
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<a href="http://www.idtdna.com/site"><img src="https://static.igem.org/mediawiki/2014/5/5e/Noko14_IDT.png"></a>
 
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<a href="http://www.cosmobio.co.jp/index_e.asp"><img src="https://static.igem.org/mediawiki/2014/e/e5/Noko14_Logo_e.jpg"></a>
 
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<a href="http://www.mbl.co.jp/e/index.html"><img src="https://static.igem.org/mediawiki/2014/4/4f/Noko14_MBL.gif"></a>
 
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Latest revision as of 17:35, 23 November 2015

　

<!DOCTYPE html>

  Pseudo Egg

　　ExTermite Coli is a new system to exterminate termites. It is composed of two parts, pseudo egg and engineered E. coli. The exterminateon of termites by ExTermite Coli involves three steps ( Figure…)：


Pseudo Egg
Termites live in dark and humid areas, often in the recess of a house, where we cannot reach. Finding termite colony is therefore very difficult.
We have to distribute our ExTermite Coli to kill termites more effectively.. One of our sponsor company, “ASANTE“, who investigates effective way to exterminate harmful insects, uses dogs to detect termites. It is an innovative way, but distribution of pesticides after finding their colony is still difficult. To produce a new system to exterminate termites, we have to deal with this problem. Our answer is…Pseudo egg.

What is Pseudo egg?
Termite is a social insect that forms colonies composed of a queen and workers. One of the main roles of the queen is the production of eggs, but the queen does not take care of her eggs or children by herself. Instead, the worker termites carry eggs, which drop out around the colony, and take care of them by licking them to moisturize and disinfect them.
Worker termites recognize their egg by its size and an egg pheromone. If they find something spherical coated by egg pheromone, they recognize it as “their egg” and they carry it []. If we make something like that, it will be recognized as their egg, and they will carry it to their colony. Therefore, by making pseudo egg containing our engineered E. coli, our E. coli will be carried into termite’s colony and distributed considering their remarkable characteristic of egg-carrying and egg-licking.
Termites recognize their egg by the following features
  • Releasing β-glucosidase and lysozyme as egg hormone
  • Spherical
  • 0.4-0.5 mm diameter
close

Worker termites also lick the eggs after carrying them into their colony. Actually, the termite’s saliva and egg pheromone contains the same substances. Licking helps to maintain the egg’s proper moisture levels and keep viruses away. Additionally, worker termites have a habit to grooming each other to exchange substances in their gut. These instincts are useful to deliver ExTermite Coli into the termite’s body.

We focused on these termite’s instincts, and we made pseudo egg.
To investigate if we can make engineered E. coli distributed using pseudo egg, we did some experiments.

＊Caution!!＊: Following pages may contain some pictures of termites.
If you dislike insects, discretion is advised when viewing the “Details” section.
maintained termites
We have maintained the following kinds of termites for our experiments.


・Formosan subterranean termite ( Coptotermes formosanus ) (イエシロアリ)

This termite cause the greatest problem to buildings. They can eat wood, paper, and can also damage concrete buildings. Their reproductive power is so strong that it can form huge colonies consisting of one or two million termites.


・Japanese subterranean termite (Reticulitermes speratus) (ヤマトシロアリ)

Japanese subterranean termite has the largest distribution in Japan.This species of termite does not have strong reproductive power relative to Formosan subterranean termite, but it is resistant to cold.


・Japanese damp-wood termite (Hodotermopsis sjostedti)(オオシロアリ)

The most characteristic feature of Japanese damp-wood termite is its small size. While the above two species are 3-5mm long, Japanese damp-wood termite is 1-1.5 mm long. This species of termite is distributed in the south area of Japan.


close

We would like to express our gratitude for ASANTE for provided these termites.



Using these three kinds of termites, we did experiments to confirm whether our pseudo eggs were carried by termites.
First, we made pseudo eggs consisting of sodium alginate, CaCl2, and LB medium. Dripping solution of sodium alginate into CaCl2 solution makes tiny beads coated by thin film of calcium alginate. LB medium added to the mixture remains inside these tiny beads.



Making Pseudo Egg
Mixture of sodium alginate (10% w/w) and LB medium was dripped into a solution of CaCl2 by cylinder (the internal diameter：0.5mm).
After making tiny beads, they were sorted by their size and 0.4 ~ 0.5 mm diameter beads were selected for experiments.
Selected beads were then coated with egg pheromone (1 µg/mL β-glucosidase and 1 µg/mL lysozyme).

close

Pseudo eggs were put on the petri dish with wet Kimtowel and nine Formosan subterranean termites released into the dish. This dish was observed for one week, and then we counted the number of the pseudo egg to check whether the pseudo eggs were moved. Fig(3) shows the result of this experiment. As a result, we find out that the number of pseudo eggs coated by pheromone was decreased compared to that of control pseudo eggs.



　Next, we checked whether the E. coli remains attached on pseudo eggs.To carry ExTermite Coli to termites by using pseudo egg, E. coli should remain attached on the surface of pseudo eggs. Also, E. coli must remain viable for a long time, until it reaches a termite’s gut. We checked these two concerns by the following experiments.
　Figure (4) shows the scheme of experiment using RFP-expressing E. coli. We made an E. coli that expresses red fluorescence protein (RFP) and mixed it with pseudo eggs. Then, we put the pseudo eggs on a LB agarose plate to check whether inside E. coli can come out from the pseudo eggs. After 24 h incubation at 37℃, we moved the pseudo eggs to the next square place. If the RFP-expressing E. coli can come out from the pseudo egg, red colony will be formed. Also, if the RFP-expressing E. coli can live on the pseudo egg, red colony will be formed on the second square place.

　From the Figure (4), we could find that the E. coli could come out from the pseudo egg. It is remarkable that E. coli can come out from inside of the pseudo egg.


　Finally, to check whether E. coli can be alive on the surface of the pseudo egg, we also made pseudo eggs which were coated by RFP-expressing E. coli and set on a LB agarose plate in the same way as above. After 24 h incubation at 37℃, we moved the pseudo eggs to the next square place. If the RFP-expressing E. coli can live on the pseudo egg, red colony will be formed on the second square place.

　　


　As a result, we found that RFP-expressing E. coli coated outside of the pseudo egg can live for two days on the surface of the pseudo egg. That means by this method of making, pseudo egg can carry E. coli for some days to distribute E. coli between termites. We are now trying to lengthen the period E. coli can survive by examining the components of pseudo egg.