http://2011.igem.org/wiki/index.php?title=Special:Contributions/Takuya_1613&feed=atom&limit=50&target=Takuya_1613&year=&month=2011.igem.org - User contributions [en]2024-03-29T07:27:42ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-29T03:51:08Z<p>Takuya 1613: </p>
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<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/25/Creating_Perception_1.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function.<br />
</p><br />
<br />
<p><br />
For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology. <br />
(<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a>) <br /><br />
<br />
The aim of the project is to make Mars, a cold and inhospitable planet, into a more habitable planet. This series consists of three parts: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. Each of the cards represents one of the three parts. When the all cards are gathered, the aim is achieved. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/5f/Igemcardgame.png" alt="iGEM card game" width="600px"/> <br />
</div><br />
<br />
<h2 id="poster">3. Posters</h2><br />
<div align="center" style="float: right;"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b7/%E5%9B%B31.png/449px-%E5%9B%B31.png" alt="iGEM poster" width="200px"/> <br />
</div><br />
<p><br />
We created three posters to explain Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. In it, the characters make bacteria which can produce delicious drink. Another poster used metaphors and many illustrations to compare combining genetic parts to make new functions with combining kitchen tools to make dishes.<br />
A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<br />
<h2 id="question" style="clear:both;">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br />
<p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fb/Age-groups.png" width="350px" /><br />
</div> <br />
</p><br />
<h3 id="4.1">4.1 All respondents</h3><br />
<p><br />
206 people answered these questions. Here is the results we obtained.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" width="600px" /><br />
</div><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
<p><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br />
</div><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
<p><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" width="600px" /><br />
</div><br />
<br />
<h3 id="4.4">4.4 What would you like to make using synthetic biology?</h3><br />
<p><br />
119 people answeres Q4 of our questionnaire.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e8/Human_Practice_Q4.png" width="600px" /><br /><br />
</div><br />
According to results, the young tended to answer our questionnaire creatively.<br />
<br />
Q4.What would you like to make using synthetic biology?<br />
A4.<br />
・bacteria glowing like a rainbow.(10 years, man)<br />
<br />
・photosynthesizing animals(14 years, man)<br />
<br />
・Neapolitan(10 years, man) <br />
<br />
<h2 id="5.">5. Conclusions</h2><br />
<p><br />
Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity! So we concluded that our human practices were really successful. We strongly believe that after about ten years, the number of iGEMers will dramatically increase.<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-29T03:22:46Z<p>Takuya 1613: </p>
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/25/Creating_Perception_1.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function.<br />
</p><br />
<br />
<p><br />
For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology. <br />
(<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a>) <br /><br />
<br />
The aim of the project is to make Mars, a cold and inhospitable planet, into a more habitable planet. This series consists of three parts: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. Each of the cards represents one of the three parts. When the all cards are gathered, the aim is achieved. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/5f/Igemcardgame.png" alt="iGEM card game" width="600px"/> <br />
</div><br />
<br />
<h2 id="poster">3. Posters</h2><br />
<div align="center" style="float: right;"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b7/%E5%9B%B31.png/449px-%E5%9B%B31.png" alt="iGEM poster" width="200px"/> <br />
</div><br />
<p><br />
We created three posters to explain Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. In it, the characters make bacteria which can produce delicious drink. Another poster used metaphors and many illustrations to compare combining genetic parts to make new functions with combining kitchen tools to make dishes.<br />
A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<br />
<h2 id="question" style="clear:both;">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br /><br />
<p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fb/Age-groups.png" width="350px" /><br /><br />
</div> <br />
</p><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h3 id="4.4">4.4 What would you like to make using synthetic biology?</h3><br />
119 people answeres Q4 of our questionnaire.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e8/Human_Practice_Q4.png" width="600px" /><br /><br />
</div><br /><br /><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity! So we concluded that our human practices were really successful. We strongly believe that after about ten years, the number of iGEMers will dramatically increase.<br />
</p><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-29T03:20:47Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#Overview">1. OverView</a></li><br />
<li><a href="#card">2. The iGEM Card Game</a></li><br />
<li><a href="#poster">3. Posters</a></li><br />
<li><br />
<a href="#question">4. Questionnaire</a><br />
<ul><br />
<li><a href="#4.1">4.1 All respondents</a></li><br />
<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
<li><a href="#4.3">4.3 Respondents over 10 years old</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/25/Creating_Perception_1.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function.<br />
</p><br />
<br />
<p><br />
For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology. <br />
(<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a>) <br /><br />
<br />
The aim of the project is to make Mars, a cold and inhospitable planet, into a more habitable planet. This series consists of three parts: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. Each of the cards represents one of the three parts. When the all cards are gathered, the aim is achieved. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/5f/Igemcardgame.png" alt="iGEM card game" width="600px"/> <br />
</div><br />
<br />
<h2 id="poster">3. Posters</h2><br />
<div align="center" style="float: right;"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b7/%E5%9B%B31.png/449px-%E5%9B%B31.png" alt="iGEM poster" width="200px"/> <br />
</div><br />
<p><br />
We created three posters to explain Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. In it, the characters make bacteria which can produce delicious drink. Another poster used metaphors and many illustrations to compare combining genetic parts to make new functions with combining kitchen tools to make dishes.<br />
A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<br />
<h2 id="question" style="clear:both;">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br /><br />
<p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fb/Age-groups.png" width="350px" /><br /><br />
</div> <br />
</p><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h3 id="4.4">4.4 What would you like to make using synthetic biology?</h3><br />
119 people answeres Q4 of our questionnaire.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e8/Human_Practice_Q4.png" width="600px" /><br /><br />
</div><br /><br /><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity! So we concluded that our human practices were really successful. We strongly believe that after about ten years, the number of iGEMers will dramatically increase.<br />
</p><br />
<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-29T03:20:29Z<p>Takuya 1613: </p>
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<li><a href="#poster">3. Posters</a></li><br />
<li><br />
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<ul><br />
<li><a href="#4.1">4.1 All respondents</a></li><br />
<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
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<!-- ############ Write main contents here ############### --><br />
<br />
<!-- page title --><br />
<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/25/Creating_Perception_1.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function.<br />
</p><br />
<br />
<p><br />
For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology. <br />
(<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a>) <br /><br />
<br />
The aim of the project is to make Mars, a cold and inhospitable planet, into a more habitable planet. This series consists of three parts: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. Each of the cards represents one of the three parts. When the all cards are gathered, the aim is achieved. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/5f/Igemcardgame.png" alt="iGEM card game" width="600px"/> <br />
</div><br />
<br />
<h2 id="poster">3. Posters</h2><br />
<div align="center" style="float: right;"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b7/%E5%9B%B31.png/449px-%E5%9B%B31.png" alt="iGEM poster" width="200px"/> <br />
</div><br />
<p><br />
We created three posters to explain Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. In it, the characters make bacteria which can produce delicious drink. Another poster used metaphors and many illustrations to compare combining genetic parts to make new functions with combining kitchen tools to make dishes.<br />
A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<br />
<h2 id="question" style="clear:both;">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br /><br />
<p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fb/Age-groups.png" width="350px" /><br /><br />
</div> <br />
</p><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h3 id="4.4">4.4 What would you like to make using synthetic biology?</h3><br />
119 people answeres Q4 of our questionnaire.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e8/Human_Practice_Q4.png" width="600px" /><br /><br />
</div><br /><br /><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity! So we concluded that our human practices were really successful. We strongly believe that after about ten years, the number of iGEMers will dramatically increase.<br />
</p><br />
<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-29T03:15:37Z<p>Takuya 1613: </p>
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<ul><br />
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<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/25/Creating_Perception_1.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function.<br />
</p><br />
<br />
<p><br />
For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology. <br />
(<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a>) <br /><br />
<br />
The aim of the project is to make Mars, a cold and inhospitable planet, into a more habitable planet. This series consists of three parts: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. Each of the cards represents one of the three parts. When the all cards are gathered, the aim is achieved. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/5f/Igemcardgame.png" alt="iGEM card game" width="600px"/> <br />
</div><br />
<br />
<h2 id="poster">3. Posters</h2><br />
<div align="center" style="float: right;"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b7/%E5%9B%B31.png/449px-%E5%9B%B31.png" alt="iGEM poster" width="200px"/> <br />
</div><br />
<p><br />
We created three posters to explain Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. In it, the characters make bacteria which can produce delicious drink. Another poster used metaphors and many illustrations to compare combining genetic parts to make new functions with combining kitchen tools to make dishes.<br />
A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<br />
<h2 id="question" style="clear:both;">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br /><br />
<p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fb/Age-groups.png" width="350px" /><br /><br />
</div> <br />
</p><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h3 id="4.4">4.4 What would respondents like to make using synthetic biology?</h3><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e8/Human_Practice_Q4.png" width="600px" /><br /><br />
</div><br /><br /><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity! So we concluded that our human practices were really successful. We strongly believe that after about ten years, the number of iGEMers will dramatically increase.<br />
</p><br />
<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/File:Human_Practice_Q4.pngFile:Human Practice Q4.png2011-10-29T03:10:43Z<p>Takuya 1613: </p>
<hr />
<div></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/dataTeam:Tokyo Tech/Projects/Urea-cooler/data2011-10-28T16:58:46Z<p>Takuya 1613: </p>
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<a href="#1.">1. Characterization of <i>rocF</i> and Arg box</a><br />
<ul> <br />
<li><a href="#1.1">1.1 Materials</a></li><br />
<li><a href="#1.2">1.2 Methods</a></li><br />
<li><a href="#1.3">1.3 Results</a></li><br />
</ul><br />
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<a href="#2.">2. Characterization of Ptrc-RBS-<i>rocF</i>-Argbox</a><br />
<ul> <br />
<li><a href="#2.1">2.1 Materials</a></li><br />
<li><a href="#2.2">2.2 Methods</a></li><br />
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<h1> Assay Method and Results </h1><br />
<br />
<h2 id="1.">1. Characterization of <i>rocF</i> and Arg box</h2><br />
<h3 id="1.1">1.1 Materials</h3><br />
<br />
<p><br />
Expression plasmids used in this study are shown in Table 1. <br /><br />
<div align="center"><br />
<table border="1"><br />
<caption><br />
Table 1. Expression plasmids used for Charcterization of <i>rocF</i> and Arg box<br />
</caption><br />
<tr><br />
<th>Designation</th><br />
<th>pSB3K3</th><br />
<th>pSB1C3</th><br />
</tr><br />
<tr><br />
<td>mock</td><br />
<td>PlacIQ</td><br />
<td>Alcohol-dehydrogenase(promoter-less)</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i></td><br />
<td>Ptrc-<i>rocF</i></td><br />
<td>Alcohol-dehydrogenase(promoter-less)</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> + Arg box</td><br />
<td>Ptrc-<i>rocF</i></td><br />
<td>Arg box</td><br />
</tr><br />
</table><br />
</div><br />
Strain MG1655 was transformed with either mock, <i>rocF</i> or <i>rocF</i> + Arg box. As shown in Table 1, <i>rocF</i> gene was introduced on pSB3K3 and Arg boxes were introduced on pSB1C3. <br />
</p><br />
<br />
<h3 id="1.2">1.2 Methods</h3><br />
<h4>1.2.1 Preparation of samples for urea concentration assay</h4><br />
<br />
<p><br />
<ol><br />
<li><br />
A single colony of cells transformed with engineered plasmids (mock, <i>rocF</i> or <i>rocF</i>+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics and grown to saturation at 37&deg;C.<br />
</li><br />
<li><br />
The saturated culture was diluted 50-fold, grown till the log phase (OD<sub>600</sub> = 0.5).<br />
</li><br />
<li><br />
The culture was induced with 1 mM IPTG at 37&deg;C for 1 hour.<br />
</li><br />
<li><br />
1.5 mL of culture was centrifuged at 9,000 rpm for 1 minute and the supernatant fluid was used as a sample for urea concentration assay.<br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h4>1.2.2 Urea concentration assay</h4><br />
<p><br />
Urea concentrations of the samples were determined colorimetrically with <a href="http://www.clonagen.com/clonagen/ab52e63f-4e38-4465-b325-5fd126415f1a/quantichrom_urea_assay_kit_product.aspx">DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems</a>.<br /><br />
Detailed methods are as follows.<br />
<ol><br />
<li><br />
10 &micro;L of the supernatant fluid from each sample, 10 &micro;L blank(LB),and 10 &micro;L standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates.<br />
</li><br />
<li><br />
200 &micro;L working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.<br />
</li><br />
<li><br />
The mixture was incubated for 20 munites at room temperature.<br />
</li><br />
<li><br />
Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as<br align="center" /><br />
<div><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Kit_equation.png" alt="equation" />.<br />
</div><br /><br />
ODSAMPLE, ODBLANK and ODSTANDARD are OD<sub>450</sub> values of sample, standard and blank, respectively. <br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h5>Linear plot of OD<sub>450</sub> vs. urea concentraion</h5><br />
<p><br />
To test linearlity between OD<sub>450</sub> vs. urea concentraion, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate with this urea assay kit in the same way as our bacterial samples. Plot of OD<sub>450</sub> values vs. urea concentraions we tested showed very good linearlity as shown in Fig. 1.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/81/Linear_plot.png" alt="Standard curve for coloring reaction in urea assay" width="400px" /><br /> <br />
fig. 1 Linear plot of OD<sub>450</sub> vs. urea concentraion<br />
</div> <br />
</p><br />
<br />
<h3 id="1.3">1.3 Results</h3><br />
<p>Each sample was assayed in duplicate urea concentration detected in each sample is shown in Table 2. fig. 2 shows the average of these 2 values.<br /><br />
</p><br />
<br />
<div align="center"><br />
<table border="2"><br />
<caption>Table 2. Urea concentrations detected in duplicated</caption> <br />
<tr><br />
<th>Colony No.</th><br />
<th>mock</th><br />
<th><i>rocF</i></th><br />
<th><i>rocF</i> + Arg box</th><br />
</tr><br />
<tr><br />
<td>#1</td><br />
<td>1.9</td><br />
<td>4.9</td><br />
<td>7.3</td><br />
</tr><br />
<tr><br />
<td>#2</td><br />
<td>0.75</td><br />
<td>4.3</td><br />
<td>7.2</td><br />
</tr><br />
<tr><br />
<td>Average</td><br />
<td>1.3</td><br />
<td>4.6</td><br />
<td>7.2</td><br />
</tr><br />
<tr><br />
<td>S.D.</td><br />
<td>0.80</td><br />
<td>0.44</td><br />
<td>0.080</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/c/cb/Arg_box_on_1C3.png" width="400px" align="center" /><br />
<div class="graph_title"><br />
fig. 2 The average of concentration values detected in duplicate<br />
</div><br />
</center><br /><br />
<br />
<h2 id="2.">2. Characterization of Ptrc-RBS-<i>rocF</i>-Argbox</h2><br />
<h3 id="2.1">2.1 Materials</h3><br />
<p>Expression plasmids used in this study are listed in table 1.</p><br />
<br />
<div align="center"><br />
<table border="3"><br />
<caption>Table 3. Expression plasmids used in this study</caption><br />
<tr><br />
<th>Designation</th><br />
<th>Parent vector</th><br />
<th>Introduced sequence(s)</th><br />
</tr><br />
<tr><br />
<td>Mock (3K3)</td><br />
<td>pSB3K3</td><br />
<td>PlacIQ</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> (3K3)</td><br />
<td>pSB3K3</i></td><br />
<td>Ptrc-<i>rocF</i></td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> + Arg box (3K3)</td><br />
<td>pSB3K3</td><br />
<td>Ptrc-<i>rocF</i>-Arg box</td><br />
</tr><br />
<tr><br />
<td>Mock (6A1)</td><br />
<td>pSB6A1</td><br />
<td><i>gfp</i> (promoter-less)</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> (6A1)</td><br />
<td>pSB6A1</i></td><br />
<td>Ptrc-<i>rocF</i></td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> + Arg box (6A1)</td><br />
<td>pSB6A1</td><br />
<td>Ptrc-<i>rocF</i>-Arg box</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<p><br />
In one experiment, MG1655 (<i>argR</i> +) and JE6852 (<i>argR</i> -) were respectively transformed with either mock(3K3), <i>rocF</i>(3K3) or <i>rocF</i>-Arg box(3K3).<br />
In another experiment, MG1655 (<i>argR</i> +) and JD24293 (<i>argR</i> -) were respectively transformed with either mock(6A1), <i>rocF</i>(6A1) or <i>rocF</i>-Arg box(6A1). Both JE6852 and JD24293 were obtained from National Institute of Genetics.<br />
</p><br />
<br />
<h3 id="2.2">2.2 Methods</h3><br />
<h4>2.2.1 Preparation of samples for urea concentration assay</h4><br />
<p><br />
<br />
<ol><br />
<li><br />
A single colony of cells transformed with engineered plasmids (mock, <i>rocF</i> or <i>rocF</i>+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics and grown to saturation at 37&deg;C.<br />
</li><br />
<li><br />
The saturated culture was diluted 50-fold, grown till the log phase (OD<sub>600</sub> = 0.5).<br />
</li><br />
<li><br />
The culture was induced with 1 mM IPTG at 37&deg;C for 1 hour.<br />
</li><br />
<li><br />
1.5 mL of culture was centrifuged at 9,000 rpm for 1 minute and the supernatant fluid was used as a sample for urea concentration assay.<br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h4>2.2.2 Urea concentration assay</h4><br />
<p><br />
Urea concentrations of the samples were determined colorimetrically with <a href="http://www.clonagen.com/clonagen/ab52e63f-4e38-4465-b325-5fd126415f1a/quantichrom_urea_assay_kit_product.aspx">DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems</a>.<br /><br />
Detailed methods are as follows.<br />
<ol><br />
<li><br />
10 &micro;L of the supernatant fluid from each sample, 10 &micro;L blank(LB),and 10 &micro;L standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates.<br />
</li><br />
<li><br />
200 &micro;L working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.<br />
</li><br />
<li><br />
The mixture was incubated for 20 munites at room temperature.<br />
</li><br />
<li><br />
Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as<br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Kit_equation.png" alt="equation" />.<br /><br />
ODSAMPLE, ODBLANK and ODSTANDARD are OD<sub>450</sub> values of sample, standard and blank, respectively. <br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h5>Linear plot of OD<sub>450</sub> vs. urea concentraion</h5><br />
<p><br />
To test linearlity between OD<sub>450</sub> vs. urea concentraion, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate with this urea assay kit in the same way as our bacterial samples. Plot of OD<sub>450</sub> values vs. urea concentraions we tested showed very good linearlity as shown in Fig. 1.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/81/Linear_plot.png" alt="Standard curve for coloring reaction in urea assay" width="400px" /><br /> <br />
fig. 3 Linear plot of OD<sub>450</sub> vs. urea concentraion<br />
</div><br />
</p><br />
<br />
<h3 id="2.3">2.3 Results</h3><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/b/bc/RocF_and_Arg_box_on_3K3.png" width="400px" align="center" /><br />
<div class="graph_title"><br />
fig. 4 Urea concentration detected in bacterial samples on pSB3K3<br />
</div><br />
</center><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/0/01/RocF_and_Arg_box_on_6A1_%283%29.png" width="400px" align="center" /><br />
<div class="graph_title"><br />
fig. 5 Urea concentration detected in bacterial samples on pSB6A1<br />
</div><br />
</center><br /><br />
<br />
<p><br />
Similar results were obtained in both the experiment with samples on pSB3K3 and the experiment with samples on pSB6A1. In MG1655(<i>argR</i> +), addition of Trc promoter-<i>rocF</i> led to production of more urea compared to mock as expected. These results show that insertion of <i>rocF</i> resulted in arginase production, therefore completing the urea cycle in <i>E. coli</i>. In the same strain, however, addition of Arg box sequence led to little change in urea production. The reason why the effect of Arg boxes was not apparent is probably that both pSB3K3 and pSB6A1 are low-copy-number plasmids. Low-copy-number plasmids are not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. Both of the plasmids containing <i>rocF</i> gene in the strains with <i>argR</i>(a gene which codes arginine repressor) loss-of-function mutant produce urea more efficiently than those in MG1655(<i>argR</i> +).<br />
</p><br />
<p><br />
These results are in line with the fact that for strains with <i>argR</i> loss-of-function mutant, deactivation of arginine repressor by Arg boxes is not needed and addition of the Arg box does not result in a significant increase of urea production. <br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/dataTeam:Tokyo Tech/Projects/Urea-cooler/data2011-10-28T16:58:20Z<p>Takuya 1613: </p>
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<ul><br />
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<a href="#1.">1. Characterization of <i>rocF</i> and Arg box</a><br />
<ul> <br />
<li><a href="#1.1">1.1 Materials</a></li><br />
<li><a href="#1.2">1.2 Methods</a></li><br />
<li><a href="#1.3">1.3 Results</a></li><br />
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<a href="#2.">2. Characterization of Ptrc-RBS-<i>rocF</i>-Argbox</a><br />
<ul> <br />
<li><a href="#2.1">2.1 Materials</a></li><br />
<li><a href="#2.2">2.2 Methods</a></li><br />
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<h1> Assay Method and Results </h1><br />
<br />
<h2 id="1.">1. Characterization of <i>rocF</i> and Arg box</h2><br />
<h3 id="1.1">1.1 Materials</h3><br />
<br />
<p><br />
Expression plasmids used in this study are shown in Table 1. <br /><br />
<div align="center"><br />
<table border="1"><br />
<caption><br />
Table 1. Expression plasmids used for Charcterization of <i>rocF</i> and Arg box<br />
</caption><br />
<tr><br />
<th>Designation</th><br />
<th>pSB3K3</th><br />
<th>pSB1C3</th><br />
</tr><br />
<tr><br />
<td>mock</td><br />
<td>PlacIQ</td><br />
<td>Alcohol-dehydrogenase(promoter-less)</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i></td><br />
<td>Ptrc-<i>rocF</i></td><br />
<td>Alcohol-dehydrogenase(promoter-less)</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> + Arg box</td><br />
<td>Ptrc-<i>rocF</i></td><br />
<td>Arg box</td><br />
</tr><br />
</table><br />
</div><br />
Strain MG1655 was transformed with either mock, <i>rocF</i> or <i>rocF</i> + Arg box. As shown in Table 1, <i>rocF</i> gene was introduced on pSB3K3 and Arg boxes were introduced on pSB1C3. <br />
</p><br />
<br />
<h3 id="1.2">1.2 Methods</h3><br />
<h4>1.2.1 Preparation of samples for urea concentration assay</h4><br />
<br />
<p><br />
<ol><br />
<li><br />
A single colony of cells transformed with engineered plasmids (mock, <i>rocF</i> or <i>rocF</i>+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics and grown to saturation at 37&deg;C.<br />
</li><br />
<li><br />
The saturated culture was diluted 50-fold, grown till the log phase (OD<sub>600</sub> = 0.5).<br />
</li><br />
<li><br />
The culture was induced with 1 mM IPTG at 37&deg;C for 1 hour.<br />
</li><br />
<li><br />
1.5 mL of culture was centrifuged at 9,000 rpm for 1 minute and the supernatant fluid was used as a sample for urea concentration assay.<br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h4>1.2.2 Urea concentration assay</h4><br />
<p><br />
Urea concentrations of the samples were determined colorimetrically with <a href="http://www.clonagen.com/clonagen/ab52e63f-4e38-4465-b325-5fd126415f1a/quantichrom_urea_assay_kit_product.aspx">DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems</a>.<br /><br />
Detailed methods are as follows.<br />
<ol><br />
<li><br />
10 &micro;L of the supernatant fluid from each sample, 10 &micro;L blank(LB),and 10 &micro;L standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates.<br />
</li><br />
<li><br />
200 &micro;L working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.<br />
</li><br />
<li><br />
The mixture was incubated for 20 munites at room temperature.<br />
</li><br />
<li><br />
Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as<br align="center" /><br />
<div><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Kit_equation.png" alt="equation" />.<br />
</div><br /><br />
ODSAMPLE, ODBLANK and ODSTANDARD are OD<sub>450</sub> values of sample, standard and blank, respectively. <br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h5>Linear plot of OD<sub>450</sub> vs. urea concentraion</h5><br />
<p><br />
To test linearlity between OD<sub>450</sub> vs. urea concentraion, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate with this urea assay kit in the same way as our bacterial samples. Plot of OD<sub>450</sub> values vs. urea concentraions we tested showed very good linearlity as shown in Fig. 1.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/81/Linear_plot.png" alt="Standard curve for coloring reaction in urea assay" width="400px" /><br /> <br />
fig. 1 Linear plot of OD<sub>450</sub> vs. urea concentraion<br />
</div> <br />
</p><br />
<br />
<h3 id="1.3">1.3 Results</h3><br />
<p>Each sample was assayed in duplicate urea concentration detected in each sample is shown in Table 2. fig. 2 shows the average of these 2 values.<br /><br />
</p><br />
<br />
<div align="center"><br />
<table border="2"><br />
<caption>Table 2. Urea concentrations detected in duplicated</caption> <br />
<tr><br />
<th>Colony No.</th><br />
<th>mock</th><br />
<th><i>rocF</i></th><br />
<th><i>rocF</i> + Arg box</th><br />
</tr><br />
<tr><br />
<td>#1</td><br />
<td>1.9</td><br />
<td>4.9</td><br />
<td>7.3</td><br />
</tr><br />
<tr><br />
<td>#2</td><br />
<td>0.75</td><br />
<td>4.3</td><br />
<td>7.2</td><br />
</tr><br />
<tr><br />
<td>Average</td><br />
<td>1.3</td><br />
<td>4.6</td><br />
<td>7.2</td><br />
</tr><br />
<tr><br />
<td>S.D.</td><br />
<td>0.80</td><br />
<td>0.44</td><br />
<td>0.080</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/c/cb/Arg_box_on_1C3.png" width="400px" align="center" /><br />
<div class="graph_title"><br />
fig. 2 The average of concentration values detected in duplicate<br />
</div><br />
</center><br /><br />
<br />
<h2 id="2.">2. Characterization of Ptrc-RBS-<i>rocF</i>-Argbox</h2><br />
<h3 id="2.1">2.1 Materials</h3><br />
<p>Expression plasmids used in this study are listed in table 1.</p><br />
<br />
<div align="center"><br />
<table border="3"><br />
<caption>Table 3. Expression plasmids used in this study</caption><br />
<tr><br />
<th>Designation</th><br />
<th>Parent vector</th><br />
<th>Introduced sequence(s)</th><br />
</tr><br />
<tr><br />
<td>Mock (3K3)</td><br />
<td>pSB3K3</td><br />
<td>PlacIQ</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> (3K3)</td><br />
<td>pSB3K3</i></td><br />
<td>Ptrc-<i>rocF</i></td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> + Arg box (3K3)</td><br />
<td>pSB3K3</td><br />
<td>Ptrc-<i>rocF</i>-Arg box</td><br />
</tr><br />
<tr><br />
<td>Mock (6A1)</td><br />
<td>pSB6A1</td><br />
<td><i>gfp</i> (promoter-less)</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> (6A1)</td><br />
<td>pSB6A1</i></td><br />
<td>Ptrc-<i>rocF</i></td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> + Arg box (6A1)</td><br />
<td>pSB6A1</td><br />
<td>Ptrc-<i>rocF</i>-Arg box</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<p><br />
In one experiment, MG1655 (<i>argR</i> +) and JE6852 (<i>argR</i> -) were respectively transformed with either mock(3K3), <i>rocF</i>(3K3) or <i>rocF</i>-Arg box(3K3).<br />
In another experiment, MG1655 (<i>argR</i> +) and JD24293 (<i>argR</i> -) were respectively transformed with either mock(6A1), <i>rocF</i>(6A1) or <i>rocF</i>-Arg box(6A1). Both JE6852 and JD24293 were obtained from National Institute of Genetics.<br />
</p><br />
<br />
<h3 id="2.2">2.2 Methods</h3><br />
<h4>2.2.1 Preparation of samples for urea concentration assay</h4><br />
<p><br />
<br />
<ol><br />
<li><br />
A single colony of cells transformed with engineered plasmids (mock, <i>rocF</i> or <i>rocF</i>+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics and grown to saturation at 37&deg;C.<br />
</li><br />
<li><br />
The saturated culture was diluted 50-fold, grown till the log phase (OD<sub>600</sub> = 0.5).<br />
</li><br />
<li><br />
The culture was induced with 1 mM IPTG at 37&deg;C for 1 hour.<br />
</li><br />
<li><br />
1.5 mL of culture was centrifuged at 9,000 rpm for 1 minute and the supernatant fluid was used as a sample for urea concentration assay.<br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h4>2.2.2 Urea concentration assay</h4><br />
<p><br />
Urea concentrations of the samples were determined colorimetrically with <a href="http://www.clonagen.com/clonagen/ab52e63f-4e38-4465-b325-5fd126415f1a/quantichrom_urea_assay_kit_product.aspx">DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems</a>.<br /><br />
Detailed methods are as follows.<br />
<ol><br />
<li><br />
10 &micro;L of the supernatant fluid from each sample, 10 &micro;L blank(LB),and 10 &micro;L standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates.<br />
</li><br />
<li><br />
200 &micro;L working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.<br />
</li><br />
<li><br />
The mixture was incubated for 20 munites at room temperature.<br />
</li><br />
<li><br />
Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as<br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Kit_equation.png" alt="equation" />.<br /><br />
ODSAMPLE, ODBLANK and ODSTANDARD are OD<sub>450</sub> values of sample, standard and blank, respectively. <br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h5>Linear plot of OD<sub>450</sub> vs. urea concentraion</h5><br />
<p><br />
To test linearlity between OD<sub>450</sub> vs. urea concentraion, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate with this urea assay kit in the same way as our bacterial samples. Plot of OD<sub>450</sub> values vs. urea concentraions we tested showed very good linearlity as shown in Fig. 1.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/81/Linear_plot.png" alt="Standard curve for coloring reaction in urea assay" width="400px" /><br /> <br />
fig. 3 Linear plot of OD<sub>450</sub> vs. urea concentraion<br />
</div><br />
</p><br />
<br />
<h3 id="2.3">2.3 Results</h3><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/b/bc/RocF_and_Arg_box_on_3K3.png" width="400px" align="center" /><br />
<div class="graph_title"><br />
fig. 4 Urea concentration detected in bacterial samples on pSB3K3<br />
</div><br />
</center><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/0/01/RocF_and_Arg_box_on_6A1_%283%29.png" width="400px" align="center" /><br />
<div class="graph_title"><br />
fig. 5 Urea concentration detected in bacterial samples on pSB6A1<br />
</div><br />
</center><br /><br />
<br />
<p><br />
Similar results were obtained in both the experiment with samples on pSB3K3 and the experiment with samples on pSB6A1. In MG1655(<i>argR</i> +), addition of Trc promoter-<i>rocF</i> led to production of more urea compared to mock as expected. These results show that insertion of <i>rocF</i> resulted in arginase production, therefore completing the urea cycle in <i>E. coli</i>. In the same strain, however, addition of Arg box sequence led to little change in urea production. The reason why the effect of Arg boxes was not apparent is probably that both pSB3K3 and pSB6A1 are low-copy-number plasmids. Low-copy-number plasmids are not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. Both of the plasmids containing <i>rocF</i> gene in the strains with <i>argR</i>(a gene which codes arginine repressor) loss-of-function mutant produce urea more efficiently than those in MG1655(<i>argR</i> +).<br />
</p><br />
<p><br />
These results are in line with the fact that for strains with <i>argR</i> loss-of-function mutant, deactivation of arginine repressor by Arg boxes is not needed and addition of the Arg box does not result in a significant increase of urea production. <br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/dataTeam:Tokyo Tech/Projects/Urea-cooler/data2011-10-28T16:56:43Z<p>Takuya 1613: </p>
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<a href="#1.">1. Characterization of <i>rocF</i> and Arg box</a><br />
<ul> <br />
<li><a href="#1.1">1.1 Materials</a></li><br />
<li><a href="#1.2">1.2 Methods</a></li><br />
<li><a href="#1.3">1.3 Results</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="#2.">2. Characterization of Ptrc-RBS-<i>rocF</i>-Argbox</a><br />
<ul> <br />
<li><a href="#2.1">2.1 Materials</a></li><br />
<li><a href="#2.2">2.2 Methods</a></li><br />
<li><a href="#2.3">2.3 Results</a></li><br />
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<h1> Assay Method and Results </h1><br />
<br />
<h2 id="1.">1. Characterization of <i>rocF</i> and Arg box</h2><br />
<h3 id="1.1">1.1 Materials</h3><br />
<br />
<p><br />
Expression plasmids used in this study are shown in Table 1. <br /><br />
<div align="center"><br />
<table border="1"><br />
<caption><br />
Table 1. Expression plasmids used for Charcterization of <i>rocF</i> and Arg box<br />
</caption><br />
<tr><br />
<th>Designation</th><br />
<th>pSB3K3</th><br />
<th>pSB1C3</th><br />
</tr><br />
<tr><br />
<td>mock</td><br />
<td>PlacIQ</td><br />
<td>Alcohol-dehydrogenase(promoter-less)</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i></td><br />
<td>Ptrc-<i>rocF</i></td><br />
<td>Alcohol-dehydrogenase(promoter-less)</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> + Arg box</td><br />
<td>Ptrc-<i>rocF</i></td><br />
<td>Arg box</td><br />
</tr><br />
</table><br />
</div><br />
Strain MG1655 was transformed with either mock, <i>rocF</i> or <i>rocF</i> + Arg box. As shown in Table 1, <i>rocF</i> gene was introduced on pSB3K3 and Arg boxes were introduced on pSB1C3. <br />
</p><br />
<br />
<h3 id="1.2">1.2 Methods</h3><br />
<h4>1.2.1 Preparation of samples for urea concentration assay</h4><br />
<br />
<p><br />
<ol><br />
<li><br />
A single colony of cells transformed with engineered plasmids (mock, <i>rocF</i> or <i>rocF</i>+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics and grown to saturation at 37&deg;C.<br />
</li><br />
<li><br />
The saturated culture was diluted 50-fold, grown till the log phase (OD<sub>600</sub> = 0.5).<br />
</li><br />
<li><br />
The culture was induced with 1 mM IPTG at 37&deg;C for 1 hour.<br />
</li><br />
<li><br />
1.5 mL of culture was centrifuged at 9,000 rpm for 1 minute and the supernatant fluid was used as a sample for urea concentration assay.<br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h4>1.2.2 Urea concentration assay</h4><br />
<p><br />
Urea concentrations of the samples were determined colorimetrically with <a href="http://www.clonagen.com/clonagen/ab52e63f-4e38-4465-b325-5fd126415f1a/quantichrom_urea_assay_kit_product.aspx">DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems</a>.<br /><br />
Detailed methods are as follows.<br />
<ol><br />
<li><br />
10 &micro;L of the supernatant fluid from each sample, 10 &micro;L blank(LB),and 10 &micro;L standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates.<br />
</li><br />
<li><br />
200 &micro;L working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.<br />
</li><br />
<li><br />
The mixture was incubated for 20 munites at room temperature.<br />
</li><br />
<li><br />
Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as<br align="center" /><br />
<div><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Kit_equation.png" alt="equation" />.<br />
</div><br /><br />
ODSAMPLE, ODBLANK and ODSTANDARD are OD<sub>450</sub> values of sample, standard and blank, respectively. <br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h5>Linear plot of OD<sub>450</sub> vs. urea concentraion</h5><br />
<p><br />
To test linearlity between OD<sub>450</sub> vs. urea concentraion, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate with this urea assay kit in the same way as our bacterial samples. Plot of OD<sub>450</sub> values vs. urea concentraions we tested showed very good linearlity as shown in Fig. 1.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/81/Linear_plot.png" alt="Standard curve for coloring reaction in urea assay" width="400px" /><br /> <br />
fig. 1 Linear plot of OD<sub>450</sub> vs. urea concentraion<br />
</div> <br />
</p><br />
<br />
<h3 id="1.3">1.3 Results</h3><br />
<p>Each sample was assayed in duplicate urea concentration detected in each sample is shown in Table 2. fig. 2 shows the average of these 2 values.<br /><br />
</p><br />
<br />
<div align="center"><br />
<table border="2"><br />
<caption>Table 2. Urea concentrations detected in duplicated</caption> <br />
<tr><br />
<th>Colony No.</th><br />
<th>mock</th><br />
<th><i>rocF</i></th><br />
<th><i>rocF</i> + Arg box</th><br />
</tr><br />
<tr><br />
<td>#1</td><br />
<td>1.9</td><br />
<td>4.9</td><br />
<td>7.3</td><br />
</tr><br />
<tr><br />
<td>#2</td><br />
<td>0.75</td><br />
<td>4.3</td><br />
<td>7.2</td><br />
</tr><br />
<tr><br />
<td>Average</td><br />
<td>1.3</td><br />
<td>4.6</td><br />
<td>7.2</td><br />
</tr><br />
<tr><br />
<td>S.D.</td><br />
<td>0.80</td><br />
<td>0.44</td><br />
<td>0.080</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/c/cb/Arg_box_on_1C3.png" width="400px" align="center" /><br />
<div class="graph_title"><br />
fig. 2 The average of concentration values detected in duplicate<br />
</div><br />
</center><br /><br />
<br />
<h2 id="2.">2. Characterization of Ptrc-RBS-<i>rocF</i>-Argbox</h2><br />
<h3 id="2.1">2.1 Materials</h3><br />
<p>Expression plasmids used in this study are listed in table 1.</p><br />
<br />
<div align="center"><br />
<table border="3"><br />
<caption>Table 3. Expression plasmids used in this study</caption><br />
<tr><br />
<th>Designation</th><br />
<th>Parent vector</th><br />
<th>Introduced sequence(s)</th><br />
</tr><br />
<tr><br />
<td>Mock (3K3)</td><br />
<td>pSB3K3</td><br />
<td>PlacIQ</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> (3K3)</td><br />
<td>pSB3K3</i></td><br />
<td>Ptrc-<i>rocF</i></td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> + Arg box (3K3)</td><br />
<td>pSB3K3</td><br />
<td>Ptrc-<i>rocF</i>-Arg box</td><br />
</tr><br />
<tr><br />
<td>Mock (6A1)</td><br />
<td>pSB6A1</td><br />
<td><i>gfp</i> (promoter-less)</td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> (6A1)</td><br />
<td>pSB6A1</i></td><br />
<td>Ptrc-<i>rocF</i></td><br />
</tr><br />
<tr><br />
<td><i>rocF</i> + Arg box (6A1)</td><br />
<td>pSB6A1</td><br />
<td>Ptrc-<i>rocF</i>-Arg box</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<p><br />
In one experiment, MG1655 (<i>argR</i> +) and JE6852 (<i>argR</i> -) were respectively transformed with either mock(3K3), <i>rocF</i>(3K3) or <i>rocF</i>-Arg box(3K3).<br />
In another experiment, MG1655 (<i>argR</i> +) and JD24293 (<i>argR</i> -) were respectively transformed with either mock(6A1), <i>rocF</i>(6A1) or <i>rocF</i>-Arg box(6A1). Both JE6852 and JD24293 were obtained from National Institute of Genetics.<br />
</p><br />
<br />
<h3 id="2.2">2.2 Methods</h3><br />
<h4>2.2.1 Preparation of samples for urea concentration assay</h4><br />
<p><br />
<br />
<ol><br />
<li><br />
A single colony of cells transformed with engineered plasmids (mock, <i>rocF</i> or <i>rocF</i>+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics and grown to saturation at 37&deg;C.<br />
</li><br />
<li><br />
The saturated culture was diluted 50-fold, grown till the log phase (OD<sub>600</sub> = 0.5).<br />
</li><br />
<li><br />
The culture was induced with 1 mM IPTG at 37&deg;C for 1 hour.<br />
</li><br />
<li><br />
1.5 mL of culture was centrifuged at 9,000 rpm for 1 minute and the supernatant fluid was used as a sample for urea concentration assay.<br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h4>2.2.2 Urea concentration assay</h4><br />
<p><br />
Urea concentrations of the samples were determined colorimetrically with <a href="http://www.clonagen.com/clonagen/ab52e63f-4e38-4465-b325-5fd126415f1a/quantichrom_urea_assay_kit_product.aspx">DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems</a>.<br /><br />
Detailed methods are as follows.<br />
<ol><br />
<li><br />
10 &micro;L of the supernatant fluid from each sample, 10 &micro;L blank(LB),and 10 &micro;L standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates.<br />
</li><br />
<li><br />
200 &micro;L working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.<br />
</li><br />
<li><br />
The mixture was incubated for 20 munites at room temperature.<br />
</li><br />
<li><br />
Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as<br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Kit_equation.png" alt="equation" />.<br /><br />
ODSAMPLE, ODBLANK and ODSTANDARD are OD<sub>450</sub> values of sample, standard and blank, respectively. <br />
</li><br />
</ol><br />
<br />
</p><br />
<br />
<h5>Linear plot of OD<sub>450</sub> vs. urea concentraion</h5><br />
<p><br />
To test linearlity between OD<sub>450</sub> vs. urea concentraion, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate with this urea assay kit in the same way as our bacterial samples. Plot of OD<sub>450</sub> values vs. urea concentraions we tested showed very good linearlity as shown in Fig. 1.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/81/Linear_plot.png" alt="Standard curve for coloring reaction in urea assay" width="400px" /><br /> <br />
fig. 3 Linear plot of OD<sub>450</sub> vs. urea concentraion<br />
</div><br />
</p><br />
<br />
<h3 id="2.3">2.3 Results</h3><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/b/bc/RocF_and_Arg_box_on_3K3.png" width="400px" align="center" /><br />
<div class="graph_title"><br />
fig. 4 Urea concentration detected in bacterial samples on pSB3K3<br />
</div><br />
</center><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/0/01/RocF_and_Arg_box_on_6A1_%283%29.png" width="400px" align="center" /><br />
<div class="graph_title"><br />
fig. 5 Urea concentration detected in bacterial samples on pSB6A1<br />
</div><br />
</center><br /><br />
<br />
<p><br />
Similar results were obtained in both the experiment with samples on pSB3K3 and the experiment with samples on pSB6A1. In MG1655(<i>argR</i> +), addition of Trc promoter-<i>rocF</i> led to production of more urea compared to mock as expected. These results show that insertion of <i>rocF</i> resulted in arginase production, therefore completing the urea cycle in <i>E. coli</i>. In the same strain, however, addition of Arg box sequence led to little change in urea production. The reason why the effect of Arg boxes was not apparent is probably that both pSB3K3 and pSB6A1 are low-copy-number plasmids. Low-copy-number plasmids are not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. Both of the plasmids containing <i>rocF</i> gene in the strains with <i>argR</i>(a gene which codes arginine repressor) loss-of-function mutant produce urea more efficiently than those in MG1655(<i>argR</i> +).<br />
</p><br />
<p><br />
These results are in line with the fact that for strains with <i>argR</i> loss-of-function mutant, deactivation of arginine repressor by Arg boxes is not needed and addition of the Arg box does not result in a significant increase of urea production. <br />
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<ul><br />
<li><a href="#intro">1.Introduction</a></li><br />
<li><a href="#Res">2.Isoprene by E.coli</a></li><br />
<li><a href="#rain">3.Aerosol by Isoprene</a></li><br />
<li><a href="#conclusion">4.conclusion</a></li><br />
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<h1> Making it Rain </h1><br />
<br />
<p><br />
<p><br />
<img src="https://static.igem.org/mediawiki/2011/e/e8/TokyoTech_rain_Illust1.png" alt="Illust" style="float:right;" width="200px" /><br />
Playing RPS with <span class="name">E. coli</span> during summer was fun, but, <br />
even if humans won, celebrations did not last long <br />
since we soon returned to complaining about the hot weather. <br />
As a prize for humans who win in our RPS game, we designed an <br />
<span class="name">E. coli</span> that can make it rain, making the hot summer more <br />
fun and refreshing (let alone applications in agriculture). <br />
</p><br />
<br />
<h2 id="intro" style="clear:both;">1. Introduction</h2><br />
<br />
<br />
<p><br />
To make it rain we focus on the substance isoprene. <br />
It has been observed that trees in tropical rainforests <br />
contribute to the formation of photo-smog aerosol in <br />
the lower atmosphere by releasing isoprene (Paulson and Seinfeld, 1992). <br />
The photo-oxidized isoprene acts as a condensation <br />
nucleus [2], may cause rain even <br />
if it is present in very low concentrations. <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/7/7c/Reaction-of-isoprene.png" alt="Fig.1" style="float:none;" width="800px" /><br />
<div class="graph_title"><br />
Fig. 1 Isoprene photo-oxidation reaction<br />
</div><br />
</div><br />
<br />
<p> <br />
It is known that the enzyme isoprene synthase can catalyze <br />
the conversion of dimethylallyl diphosphate(DMAPP) to <br />
isoprene. DMAPP is normally synthesized by <br />
<span class="name">E. coli</span>, so the only thing we need <br />
to make our bacteria synthetize isoprene is isoprene synthase. <br />
The isoprene synthase coding gene (<span class="gene">ispS</span>) <br />
is isolated from the tree poplar (Barbara Miller <i>et al</i>., 2001). <br />
<span class="name">E. coli</span> introduced this gene <br />
released isoprene into the air by diffusion [1] <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/TokyoTech_rain_fig2.png" width="658px" alt="Fig.3" /><br />
<div class="graph_title"><br />
Fig. 2 Formation of isoprene is catalyzed by isoprene synthase<br />
</div><br />
</div><br />
<br />
<p><br />
In this study, we made <span class="name">E. coli</span> <br />
synthesize isoprene by introducing <span class="gene">ispS</span>.<br />
</p><br />
<br />
<h2 id="Res">2. Isoprene by <span class="name">E. coli</span></h2><br />
<br />
<p><br />
To measure the amount of isoprene produced by <span class="name">E. coli</span> with the introduction of <span class="gene">ispS</span>, we constructed negative control PlacIQ <br />
and sample PlacIQ-RBS-<span class="gene">ispS</span>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K649303">BBa_K649303</a>)<br />
, using the <br />
PlacIQ promoter (BBa_I14032) and <span class="gene">ispS</span>. <br />
Gene <span class="gene">ispS</span> is extracted from the pMK <br />
backbone vector.<br />
(<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#Const">see more about our constructions</a>)<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/08/Rain-const.png" alt="Fig.3" width="500px" style="float:none;" /><br />
<div class="graph_title"><br />
Fig. 3 Constructions of PlacIQ and PlacIQ-RBS-<span class="gene">ispS</span><br />
</div><br />
</div><br />
<br />
<p><br />
We used Gas Chromotrography-Mass Spectrometry (GC-MS)<br />
to measure the amount of isoprene produced by <br />
<span class="name">E. coli</span>. When using GC-MS, we firstly injected a series of chloroform-diluted <br />
liquid isoprene to draw the calibration curve. Then the peaks of negative control(PlacIQ) and sample(PlacIQ-RBS-ispS) were detected at the retention time at 1.1 min. This is same to the retention time of the peak of reference material isoprene. Therefore, we concluded that our E.coli was producing isoprene as we expected.<br />
</p><br />
<p><br />
According to the calibration curve and peak areas, we calculated the isoprene produced by our <span class="name">E. coli</span> BL21 (DE3) with the introduction of <span class="name">ispS</span> is about 4.1×10<sup>-5</sup> mg/L, while negative control (PlacIQ) only produced one eighth of the sample.<br />
(<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#AP">see more about GC-MS</a>)<br />
</p><br />
<p><br />
<div align="center"><br />
(a)<br />
<a href="https://static.igem.org/mediawiki/2011/a/a1/GS-MG_assay.png"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a1/GS-MG_assay.png" width="200px"/></a><br />
(b)<br />
<img src="https://static.igem.org/mediawiki/2011/e/e4/Rain-fig4-2.jpg" alt="isprene-graph" width="400px" /><br />
</div><br />
<center>Fig. 4 isoprene detected by GC-MS (This work is done by Yuto Sugiuchi.)<br /><br />
(a)a-1:negative control(PlacIQ), a-2:sample(PlacIQ-RBS-<span class="gene">ispS</span>), a-3:reference material <br />(b)The amount of isoprene detected in <span class="name">E. coli</span> extract. </center><br />
</p> <br />
<br />
<br />
<h2 id="rain">3. Discussion</h2><br />
<p><br />
The reaction between isoprene and ozone has been studied to examine physical and chemical characteristics of the secondary organic aerosol formed. Aerosols is suspension of solid particles or liquid droplets in gas. The most common aerosol in the atmosphere are clouds, which normally consist of suspensions of water droplets or ice particles of greater density, and can later cause rain. According to those information, we designed an easy indoor experiment of reaction between isoprene and ozone, and confirmed that isoprene can make aerosol.The ozone-isoprene reaction was carried out in teflon bags as follows. To facilitate the reaction, ultraviolet radiation was used. 20 mins after the reaction started, formation of aerosol was confirmed as shown the photos below.<br />
<center><br />
<table><br />
<tr><br />
<th>Isoprene -</th><br />
<th>Isoprene +</th><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/93/Aerosol2.png" alt="aerosol2" /><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/archive/c/c0/20111027081411%21Aerosol1.png" alt="aerosol1" /><br />
</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<center>Fig. 7 aerosol conformation</center><br />
<br />
<p><br />
The picture on the left shows that when isoprene was not present no aerosol was detected even when air, water and ozone were put together under reaction conditions. On the other hand, the picture on the right shows that when isoprene was used, it formed an aerosol (this became evident because the trajectory of the laser light was visible).<br />
</p> <br />
<br />
<br/><br />
<p><br />
All in all, we confirmed that <span class="name">E. coli</span> with the insertion of <span class="gene">ispS</span> synthesizes isoprene and that isoprene makes aerosol. So our <span class="name">E. coli</span> will make it rain! We also thoroughly concerned about the safety that might come up with the using of isoprene, <a href="https://2011.igem.org/Team:Tokyo_Tech/Safety">details can be seen here.</a> <br/></p><br />
<br />
<br />
<div style="margin: 5px;"><br />
<h2>Reference </h2><br />
[1] Yaru zhao, <i>et al.</i>, Biosynthesis of isoprene in <span class="name">Escherichia coli</span> via methylerythritol phosphate (MEP) pathway, Appl Microbiol Biothechnol(2011) 90:1915-1922<br /><br />
<br />
[2] Leonardo Silva Santos, <i>et al.</i>, Mimicking the atmospheric OH-radical-mediated photooxidation of isoprene: formation of cloud-condensation nuclei polyols monitored by electrospray ionization mass spectrometry, Rapid Communication in Mass Spectrometry, 2006<br /><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/index.htmTeam:Tokyo Tech/Projects/making-rain/index.htm2011-10-28T16:46:01Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#intro">1.Introduction</a></li><br />
<li><a href="#Res">2.Isoprene by E.coli</a></li><br />
<li><a href="#rain">3.Aerosol by Isoprene</a></li><br />
<li><a href="#conclusion">4.conclusion</a></li><br />
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<h1> Making it Rain </h1><br />
<br />
<p><br />
<p><br />
<img src="https://static.igem.org/mediawiki/2011/e/e8/TokyoTech_rain_Illust1.png" alt="Illust" style="float:right;" width="200px" /><br />
Playing RPS with <span class="name">E. coli</span> during summer was fun, but, <br />
even if humans won, celebrations did not last long <br />
since we soon returned to complaining about the hot weather. <br />
As a prize for humans who win in our RPS game, we designed an <br />
<span class="name">E. coli</span> that can make it rain, making the hot summer more <br />
fun and refreshing (let alone applications in agriculture). <br />
</p><br />
<br />
<h2 id="intro" style="clear:both;">1. Introduction</h2><br />
<br />
<br />
<p><br />
To make it rain we focus on the substance isoprene. <br />
It has been observed that trees in tropical rainforests <br />
contribute to the formation of photo-smog aerosol in <br />
the lower atmosphere by releasing isoprene (Paulson and Seinfeld, 1992). <br />
The photo-oxidized isoprene acts as a condensation <br />
nucleus [2], may cause rain even <br />
if it is present in very low concentrations. <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/7/7c/Reaction-of-isoprene.png" alt="Fig.1" style="float:none;" width="800px" /><br />
<div class="graph_title"><br />
Fig. 1 Isoprene photo-oxidation reaction<br />
</div><br />
</div><br />
<br />
<p> <br />
It is known that the enzyme isoprene synthase can catalyze <br />
the conversion of dimethylallyl diphosphate(DMAPP) to <br />
isoprene. DMAPP is normally synthesized by <br />
<span class="name">E. coli</span>, so the only thing we need <br />
to make our bacteria synthetize isoprene is isoprene synthase. <br />
The isoprene synthase coding gene (<span class="gene">ispS</span>) <br />
is isolated from the tree poplar (Barbara Miller <i>et al</i>., 2001). <br />
<span class="name">E. coli</span> introduced this gene <br />
released isoprene into the air by diffusion [1] <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/TokyoTech_rain_fig2.png" width="658px" alt="Fig.3" /><br />
<div class="graph_title"><br />
Fig. 2 Formation of isoprene is catalyzed by isoprene synthase<br />
</div><br />
</div><br />
<br />
<p><br />
In this study, we made <span class="name">E. coli</span> <br />
synthesize isoprene by introducing <span class="gene">ispS</span>.<br />
</p><br />
<br />
<h2 id="Res">2. Isoprene by <span class="name">E. coli</span></h2><br />
<br />
<p><br />
To measure the amount of isoprene produced by <span class="name">E. coli</span> with the introduction of <span class="gene">ispS</span>, we constructed negative control PlacIQ <br />
and sample PlacIQ-RBS-<span class="gene">ispS</span>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K649303">BBa_K649303</a>)<br />
, using the <br />
PlacIQ promoter (BBa_I14032) and <span class="gene">ispS</span>. <br />
Gene <span class="gene">ispS</span> is extracted from the pMK <br />
backbone vector.<br />
(<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#Const">see more about our constructions</a>)<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/08/Rain-const.png" alt="Fig.3" width="500px" style="float:none;" /><br />
<div class="graph_title"><br />
Fig. 3 Constructions of PlacIQ and PlacIQ-RBS-<span class="gene">ispS</span><br />
</div><br />
</div><br />
<br />
<p><br />
We used Gas Chromotrography-Mass Spectrometry (GC-MS)<br />
to measure the amount of isoprene produced by <br />
<span class="name">E. coli</span>. When using GC-MS, we firstly injected a series of chloroform-diluted <br />
liquid isoprene to draw the calibration curve. Then the peaks of negative control(PlacIQ) and sample(PlacIQ-RBS-ispS) were detected at the retention time at 1.1 min. This is same to the retention time of the peak of reference material isoprene. Therefore, we concluded that our E.coli was producing isoprene as we expected.<br />
</p><br />
<p><br />
According to the calibration curve and peak areas, we calculated the isoprene produced by our <span class="name">E. coli</span> BL21 (DE3) with the introduction of <span class="name">ispS</span> is about 4.1×10<sup>-5</sup> mg/L, while negative control (PlacIQ) only produced one eighth of the sample.<br />
(<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#AP">see more about GC-MS</a>)<br />
</p><br />
<p><br />
<div align="center"><br />
(a)<br />
<a href="https://static.igem.org/mediawiki/2011/a/a1/GS-MG_assay.png"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a1/GS-MG_assay.png" width="200px"/></a><br />
(b)<br />
<img src="https://static.igem.org/mediawiki/2011/e/e4/Rain-fig4-2.jpg" alt="isprene-graph" width="400px" /><br />
</div><br />
<center>Fig. 4 isoprene detected by GC-MS (This work is done by Yuto Sugiuchi.)<br /><br />
(a)a-1:negative control(PlacIQ), a-2:sample(PlacIQ-RBS-<span class="gene">ispS</span>), a-3:reference material <br />(b)The amount of isoprene detected in <span class="name">E. coli</span> extract. </center><br />
</p> <br />
<br />
<br />
<h2 id="rain">3. Discussion</h2><br />
<p><br />
The reaction between isoprene and ozone has been studied to examine physical and chemical characteristics of the secondary organic aerosol formed. Aerosols is suspension of solid particles or liquid droplets in gas. The most common aerosol in the atmosphere are clouds, which normally consist of suspensions of water droplets or ice particles of greater density, and can later cause rain. According to those information, we designed an easy indoor experiment of reaction between isoprene and ozone, and confirmed that isoprene can make aerosol.The ozone-isoprene reaction was carried out in teflon bags as follows. To facilitate the reaction, ultraviolet radiation was used. 20 mins after the reaction started, formation of aerosol was confirmed as shown the photos below.<br />
<center><br />
<table><br />
<tr><br />
<th>Isoprene -</th><br />
<th>Isoprene +</th><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/93/Aerosol2.png" alt="aerosol2" /><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/archive/c/c0/20111027081411%21Aerosol1.png" alt="aerosol1" /><br />
</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<center>Fig. 7 aerosol conformation</center><br />
<br />
<p><br />
The picture on the left shows that when isoprene was not present no aerosol was detected even when air, water and ozone were put together under reaction conditions. On the other hand, the picture on the right shows that when isoprene was used, it formed an aerosol (this became evident because the trajectory of the laser light was visible).<br />
</p> <br />
<br />
<br/><br />
<p><br />
All in all, we confirmed that <span class="name">E. coli</span> with the insertion of <span class="gene">ispS</span> synthesizes isoprene and that isoprene makes aerosol. So our <span class="name">E. coli</span> will make it rain! We also thoroughly concerned about the safety that might come up with the using of isoprene, <a href="https://2011.igem.org/Team:Tokyo_Tech/Safety">details can be seen here.</a> <br/></p><br />
<br />
<br />
<div style="margin: 5px;"><br />
<h2>Reference </h2><br />
[1] Yaru zhao, <i>et al.</i>, Biosynthesis of isoprene in <span class="name">Escherichia coli</span> via methylerythritol phosphate (MEP) pathway, Appl Microbiol Biothechnol(2011) 90:1915-1922<br /><br />
<br />
[2] Leonardo Silva Santos, <i>et al.</i>, Mimicking the atmospheric OH-radical-mediated photooxidation of isoprene: formation of cloud-condensation nuclei polyols monitored by electrospray ionization mass spectrometry, Rapid Communication in Mass Spectrometry, 2006<br /><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/index.htmTeam:Tokyo Tech/Projects/making-rain/index.htm2011-10-28T13:13:36Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#intro">1.Introduction</a></li><br />
<li><a href="#Res">2.Isoprene by E.coli</a></li><br />
<li><a href="#rain">3.Aerosol by Isoprene</a></li><br />
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<h1> Making it Rain </h1><br />
<br />
<p><br />
<p><br />
<img src="https://static.igem.org/mediawiki/2011/e/e8/TokyoTech_rain_Illust1.png" alt="Illust" style="float:right;" width="200px" /><br />
Playing RPS with <span class="name">E. coli</span> during summer was fun, but, <br />
even if humans won, celebrations did not last long <br />
since we soon returned to complaining about the hot weather. <br />
As a prize for humans who win in our RPS game, we designed an <br />
<span class="name">E. coli</span> that can make it rain, making the hot summer more <br />
fun and refreshing (let alone applications in agriculture). <br />
</p><br />
<br />
<h2 id="intro" style="clear:both;">1. Introduction</h2><br />
<br />
<br />
<p><br />
To make it rain we focus on the substance isoprene. <br />
It has been observed that trees in tropical rainforests <br />
contribute to the formation of photo-smog aerosol in <br />
the lower atmosphere by releasing isoprene (Paulson and Seinfeld, 1992). <br />
The photo-oxidized isoprene acts as a condensation <br />
nucleus [2], may cause rain even <br />
if it is present in very low concentrations. <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/7/7c/Reaction-of-isoprene.png" alt="Fig.1" style="float:none;" width="800px" /><br />
<div class="graph_title"><br />
Fig. 1 Isoprene photo-oxidation reaction<br />
</div><br />
</div><br />
<br />
<p> <br />
It is known that the enzyme isoprene synthase can catalyze <br />
the conversion of dimethylallyl diphosphate(DMAPP) to <br />
isoprene. DMAPP is normally synthesized by <br />
<span class="name">E. coli</span>, so the only thing we need <br />
to make our bacteria synthetize isoprene is isoprene synthase. <br />
The isoprene synthase coding gene (<span class="gene">ispS</span>) <br />
is isolated from the tree poplar (Barbara Miller <i>et al</i>., 2001). <br />
<span class="name">E. coli</span> introduced this gene <br />
released isoprene into the air by diffusion [1] <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/TokyoTech_rain_fig2.png" width="658px" alt="Fig.3" /><br />
<div class="graph_title"><br />
Fig. 2 Formation of isoprene is catalyzed by isoprene synthase<br />
</div><br />
</div><br />
<br />
<p><br />
In this study, we made <span class="name">E. coli</span> <br />
synthesize isoprene by introducing <span class="gene">ispS</span>.<br />
</p><br />
<br />
<h2 id="Res">2. Isoprene by <span class="name">E. coli</span></h2><br />
<br />
<p><br />
To measure the amount of isoprene produced by <span class="name">E. coli</span> with the introduction of <span class="gene">ispS</span>, we constructed negative control PlacIQ <br />
and sample PlacIQ-rbs-<span class="gene">ispS</span>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K649303">BBa_K649303</a>)<br />
, using the <br />
PlacIQ promoter (BBa_I14032) and <span class="gene">ispS</span>. <br />
Gene <span class="gene">ispS</span> is extracted from the pMK <br />
backbone vector.<br />
(<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#Const">see more about our constructions</a>)<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/08/Rain-const.png" alt="Fig.3" width="500px" style="float:none;" /><br />
<div class="graph_title"><br />
Fig. 3 Constructions of PlacIQ and PlacIQ-rbs-<span class="gene">ispS</span><br />
</div><br />
</div><br />
<br />
<p><br />
We used Gas Chromotrography-Mass Spectrometry (GC-MS)<br />
to measure the amount of isoprene from <br />
<span class="name">E. coli</span>. When using GC-MS, we injected a series of chloroform-diluted <br />
liquid isoprene to draw the calibration curve. <br />
To confirm that liquid isoprene produced by <span class="name">E. coli</span> <br />
would be released as a gas, we diluted liquid isoprene in water and <br />
also in LB medium. In both cases, we could confirm isoprene was evaporated <br />
into the air.<br />
</p><br />
<p><br />
According to the calibration curve, we detected 4.1×10<sup>-5</sup> mg/L isoprene produced by <span class="name">E. coli</span> BL21 (DE3) introduced isoprene synthase, while negative control (PlacIQ) produced one eighth of our new <span class="name">E. coli</span>.<br />
(<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#AP">see more about GC-MS</a>)<br />
</p><br />
<p><br />
<div align="center"><br />
(a)<br />
<a href="https://static.igem.org/mediawiki/2011/a/a4/GS-CG_assay.png"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/GS-CG_assay.png" width="200px"/></a><br />
(b)<br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Rain-fig4-2.png" alt="isprene-graph" width="400px" /><br />
</div><br />
<center>Fig. 4 isoprene detected by GC-MS (This work is done by Yuto Sugiuchi.)<br /><br />
(a)a-1:negative control(PlacIQ), a-2:sample(PlacIQ-rbs-<span class="gene">ispS</span>), a-3:reference material <br />(b)The amount of isoprene detected in <span class="name">E. coli</span> extract. </center><br />
</p> <br />
<br />
<br />
<h2 id="rain">3.Discussion</h2><br />
<p><br />
The reaction between isoprene and ozone has been studied to examine physical and chemical characteristics of the secondary organic aerosol formed. Aerosols is suspension of solid particles or liquid droplets in gas. The most common aerosol in the atmosphere are clouds, which normally consist of suspensions of water droplets or ice particles of greater density, and can later cause rain. According to those information, we designed an easy indoor experiment of reaction between isoprene and ozone, and confirmed that isoprene can make aerosol. (<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#rain">see more...</a>)<br />
All in all, we confirmed that <span class="name">E. coli</span> with the insertion of <span class="gene">ispS</span> synthesizes isoprene and that isoprene makes aerosol. So our <span class="name">E. coli</span> will make it rain! <br/></p><br />
<p>At the same time we thoroughly concerned about the safety that might come up with the using of isoprene, <a href="https://2011.igem.org/Team:Tokyo_Tech/Safety">details can be seen here.</a><br />
<br />
</p><br />
<br />
<div style="margin: 5px;"><br />
<b>Reference </b><br /><br />
[1] Yaru zhao, <i>et al.</i>, Biosynthesis of isoprene in <span class="name">Escherichia coli</span> via methylerythritol phosphate (MEP) pathway, Appl Microbiol Biothechnol(2011) 90:1915-1922<br /><br />
<br />
[2] Leonardo Silva Santos, <i>et al.</i>, Mimicking the atmospheric OH-radical-mediated photooxidation of isoprene: formation of cloud-condensation nuclei polyols monitored by electrospray ionization mass spectrometry, Rapid Communication in Mass Spectrometry, 2006<br /><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/index.htmTeam:Tokyo Tech/Projects/making-rain/index.htm2011-10-28T13:06:28Z<p>Takuya 1613: </p>
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<li><a href="#Res">2.Isoprene by E.coli</a></li><br />
<li><a href="#rain">3.Aerosol by Isoprene</a></li><br />
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<h1> Making it Rain </h1><br />
<br />
<p><br />
<p><br />
<img src="https://static.igem.org/mediawiki/2011/e/e8/TokyoTech_rain_Illust1.png" alt="Illust" style="float:right;" width="200px" /><br />
Playing RPS with <span class="name">E. coli</span> during summer was fun, but, <br />
even if humans won, celebrations did not last long <br />
since we soon returned to complaining about the hot weather. <br />
As a prize for humans who win in our RPS game, we designed an <br />
<span class="name">E. coli</span> that can make it rain, making the hot summer more <br />
fun and refreshing (let alone applications in agriculture). <br />
</p><br />
<br />
<h2 id="intro" style="clear:both;">1. Introduction</h2><br />
<br />
<br />
<p><br />
To make it rain we focus on the substance isoprene. <br />
It has been observed that trees in tropical rainforests <br />
contribute to the formation of photo-smog aerosol in <br />
the lower atmosphere by releasing isoprene (Paulson and Seinfeld, 1992). <br />
The photo-oxidized isoprene acts as a condensation <br />
nucleus [2], may cause rain even <br />
if it is present in very low concentrations. <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/7/7c/Reaction-of-isoprene.png" alt="Fig.1" style="float:none;" width="800px" /><br />
<div class="graph_title"><br />
Fig. 1 Isoprene photo-oxidation reaction<br />
</div><br />
</div><br />
<br />
<p> <br />
It is known that the enzyme isoprene synthase can catalyze <br />
the conversion of dimethylallyl diphosphate(DMAPP) to <br />
isoprene. DMAPP is normally synthesized by <br />
<span class="name">E. coli</span>, so the only thing we need <br />
to make our bacteria synthetize isoprene is isoprene synthase. <br />
The isoprene synthase coding gene (<span class="gene">ispS</span>) <br />
is isolated from the tree poplar (Barbara Miller <i>et al</i>., 2001). <br />
<span class="name">E. coli</span> introduced this gene <br />
released isoprene into the air by diffusion [1] <br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/cd/TokyoTech_rain_fig2.png" width="658px" alt="Fig.3" /><br />
<div class="graph_title"><br />
Fig. 2 Formation of isoprene is catalyzed by isoprene synthase<br />
</div><br />
</div><br />
<br />
<p><br />
In this study, we made <span class="name">E. coli</span> <br />
synthesize isoprene by introducing <span class="gene">ispS</span>.<br />
</p><br />
<br />
<h2 id="Res">2. Isoprene by <span class="name">E. coli</span></h2><br />
<br />
<p><br />
To measure the amount of isoprene produced by <span class="name">E. coli</span> with the introduction of <span class="gene">ispS</span>, we constructed negative control PlacIQ <br />
and sample PlacIQ-rbs-<span class="gene">ispS</span>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K649303">BBa_K649303</a>)<br />
, using the <br />
PlacIQ promoter (BBa_I14032) and <span class="gene">ispS</span>. <br />
Gene <span class="gene">ispS</span> is extracted from the pMK <br />
backbone vector.<br />
(<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#Const">see more about our constructions</a>)<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/08/Rain-const.png" alt="Fig.3" width="500px" style="float:none;" /><br />
<div class="graph_title"><br />
Fig. 3 Constructions of PlacIQ and PlacIQ-rbs-<span class="gene">ispS</span><br />
</div><br />
</div><br />
<br />
<p><br />
We used Gas Chromotrography-Mass Spectrometry (GC-MS)<br />
to measure the amount of isoprene from <br />
<span class="name">E. coli</span>. When using GC-MS, we injected a series of chloroform-diluted <br />
liquid isoprene to draw the calibration curve. <br />
To confirm that liquid isoprene produced by <span class="name">E. coli</span> <br />
would be released as a gas, we diluted liquid isoprene in water and <br />
also in LB medium. In both cases, we could confirm isoprene was evaporated <br />
into the air.<br />
</p><br />
<p><br />
According to the calibration curve, we detected 4.1×10<sup>-5</sup> mg/L isoprene produced by <span class="name">E. coli</span> BL21 (DE3) introduced isoprene synthase, while negative control (PlacIQ) produced one eighth of our new <span class="name">E. coli</span>.<br />
(<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#AP">see more about GC-MS</a>)<br />
</p><br />
<p><br />
<div align="center"><br />
(a)<br />
<a href="https://static.igem.org/mediawiki/2011/a/a4/GS-CG_assay.png"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/GS-CG_assay.png" width="200px"/></a><br />
(b)<br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Rain-fig4-2.png" alt="isprene-graph" width="400px" /><br />
</div><br />
<center>Fig. 4 isoprene detected by GC-MS (This work is done by Yuto Sugiuchi.)<br /><br />
(a)a-1:negative control(PlacIQ), a-2:sample(PlacIQ-rbs-<span class="gene">ispS</span>), a-3:reference material <br />(b)The amount of isoprene detected in <span class="name">E. coli</span> extract. </center><br />
</p> <br />
<br />
<br />
<h2 id="rain">3.Discussion</h2><br />
<p><br />
The reaction between isoprene and ozone has been studied to examine physical and chemical characteristics of the secondary organic aerosol formed. Aerosols is suspension of solid particles or liquid droplets in gas. The most common aerosol in the atmosphere are clouds, which normally consist of suspensions of water droplets or ice particles of greater density, and can later cause rain. According to those information, we designed an easy indoor experiment of reaction between isoprene and ozone, and confirmed that isoprene can make aerosol. (<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/GC-Assay#rain">see more...</a>)<br />
All in all, we confirmed that <span class="name">E. coli</span> with the insertion of <span class="gene">ispS</span> synthesizes isoprene and that isoprene makes aerosol. So our <span class="name">E. coli</span> will make it rain! <br/></p><br />
<p>At the same time we thoroughly concerned about the safety that might come up with the using of isoprene, <a href="https://2011.igem.org/Team:Tokyo_Tech/Safety">details can be seen here.</a><br />
<br />
</p><br />
<br />
<div style="margin: 5px;"><br />
<b>Reference </b><br /><br />
[1] Yaru zhao, <i>et al.</i>, Biosynthesis of isoprene in <span class="name">Escherichia coli</span> via methylerythritol phosphate (MEP) pathway, Appl Microbiol Biothechnol(2011) 90:1915-1922<br /><br />
<br />
[2] Leonardo Silva Santos, <i>et al.</i>, Mimicking the atmospheric OH-radical-mediated photooxidation of isoprene: formation of cloud-condensation nuclei polyols monitored by electrospray ionization mass spectrometry, Rapid Communication in Mass Spectrometry, 2006<br /><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Modeling/RPS-game/RPS-gameTeam:Tokyo Tech/Modeling/RPS-game/RPS-game2011-10-28T10:51:30Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#1.1">1. Introduction</a></li><br />
<li><a href="#1.2">2. Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#1.3">3. How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#1.4">4. The Old Model</a></li><br />
<li><a href="#1.5">5. Our New Model</a></li><br />
<li><a href="#1.6">6. The Biological Meaning of our Model</a></li><br />
<li><a href="#1.7">7. Making it Obvious</a></li><br />
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<h1 id="1"> Survival of One Strain</h1><br />
<br />
<p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" width="500px" /><br />
</div><br />
<br />
<h2 id="1.1">1. Introduction: Minimal differences determine who will survive</h2><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a <br />
randomizer that can be used in our Rock-Paper-Scissors game, due to the fact that only one <br />
of the rival strains will survive. More specifically, we assign to each of the three <br />
rival strains either of Rock, Paper or Scissors, make them compete for survival and take <br />
the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h2 id="1.2">2. Adjusting the Model to create a True Randomizer</h2><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in 1996 by Durrett and Levin. <br />
In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. However, <br />
the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive (although it <br />
can dominate the system, i.e. have the highest population density, for definite periods <br />
of time). We will discuss more on the limitations we found in this model to be adopted as <br />
a randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h2 id="1.3">3. How the Three Types of Bacteria Compete for Survival</h2><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to <br />
outcompete their rivals: the production of a toxin (a bacteriocin called colicin)<br />
that is toxic to other strains, resistance to the toxin produced by other strains, <br />
and a higher birth rate than their rival strains. Namely, the three types <br />
of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) and colicin-sensitive <br />
<span class="name">E. coli</span> (S). The colicin-producer outcompetes the <br />
colicin-sensitive by producing the colicin. The colicin-sensitive bacteria <br />
outcompetes the colicin-resistant because its birth rate is higher <br />
than that of the colicin-resistant. The colicin-resistant outcompetes the <br />
colicin producer because its birth rate is higher than that of the colicin producer. <br />
The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" width="500px" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<br />
<h2 id="1.4">4. The Old Model</h2><br />
<br />
<p><br />
In the model described by Durrett and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). However, as will be explained afterwards, this results in a loss of balance that does not allow building a true randomizing system.<br />
</p><br />
<br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durrett and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
</div><br />
<br />
<h2 id="1.5">5. Our New Model</h2><br />
<p><br />
As mentioned before, the model proposed by Durrett and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it a <br />
biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive by <br />
outcompeting the other two strains, which will die. More specifically, we limited the resistance of the colicin-resistant <br />
bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and to the sensitive strain, <br />
and additionally the resistant strain would also be vulnerable to the colicin produced by the colicin-producer. <br />
Since which strain will be the one that survives is determined by very small differences in the initial concentrations of <br />
the three different populations of bacteria, in practice this systems becomes a randomizer because of the imprecisions in <br />
the measurements that result, for example, when using micropipettes. This randomizer describes a new competition dynamic <br />
that could not be reproduced in the previous model proposed by Durrett and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p>If we set the parameters as follows</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, we get a graph which clearly shows there are stable points on each of the three axes (Figure 1, Up).<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations we have set <br />
all of the three strains may ultimately survive for infinite peiriods of time. The differences between <br />
our model and the model of Durrett and Levin can be seen graphically in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail <br />
conditions of the model proposed by Durrett and Levin (indicated in black font)<br /><br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="1.6">6. The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its<br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in <br />
the production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h2 id="1.7">7. Making it Obvious</h2><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are <br />
paths that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this <br />
paths all have an approximately common origin. In this section we would like to show <br />
that the origin of these paths is practically the same, and that in that sense we have <br />
designed a true randomizer (since, as mentioned before, the imprecisions that result <br />
in the experimental measurements will make it impossible to make the initial<br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three different <br />
strains of <span class="name">E. coli</span> to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<br />
<p><br />
We modeled our results using Matlab. As can be seen in the graphs below, each of the strains <br />
can survive if their initial density in only tree hundredths (a.u.) greater than <br />
the other two strains' initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
</p><br />
<br/><br />
<br/><br />
<b>Reference </b><br /><br />
<p>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171.</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Modeling/RPS-game/RPS-gameTeam:Tokyo Tech/Modeling/RPS-game/RPS-game2011-10-28T10:51:10Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#1.1">1. Introduction</a></li><br />
<li><a href="#1.2">2. Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#1.3">3. How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#1.4">4. The Old Model</a></li><br />
<li><a href="#1.5">5. Our New Model</a></li><br />
<li><a href="#1.6">6. The Biological Meaning of our Model</a></li><br />
<li><a href="#1.7">7. Making it Obvious</a></li><br />
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<h1 id="1"> Survival of One Strain</h1><br />
<br />
<p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" width="500px" /><br />
</div><br />
<br />
<h2 id="1.1">1. Introduction: Minimal differences determine who will survive</h2><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a <br />
randomizer that can be used in our Rock-Paper-Scissors game, due to the fact that only one <br />
of the rival strains will survive. More specifically, we assign to each of the three <br />
rival strains either of Rock, Paper or Scissors, make them compete for survival and take <br />
the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h2 id="1.2">2. Adjusting the Model to create a True Randomizer</h2><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in 1996 by Durrett and Levin. <br />
In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. However, <br />
the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive (although it <br />
can dominate the system, i.e. have the highest population density, for definite periods <br />
of time). We will discuss more on the limitations we found in this model to be adopted as <br />
a randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h2 id="1.3">3. How the Three Types of Bacteria Compete for Survival</h2><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to <br />
outcompete their rivals: the production of a toxin (a bacteriocin called colicin)<br />
that is toxic to other strains, resistance to the toxin produced by other strains, <br />
and a higher birth rate than their rival strains. Namely, the three types <br />
of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) and colicin-sensitive <br />
<span class="name">E. coli</span> (S). The colicin-producer outcompetes the <br />
colicin-sensitive by producing the colicin. The colicin-sensitive bacteria <br />
outcompetes the colicin-resistant because its birth rate is higher <br />
than that of the colicin-resistant. The colicin-resistant outcompetes the <br />
colicin producer because its birth rate is higher than that of the colicin producer. <br />
The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" width="500px" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<br />
<h2 id="1.4">4. The Old Model</h2><br />
<br />
<p><br />
In the model described by Durrett and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). However, as will be explained afterwards, this results in a loss of balance that does not allow building a true randomizing system.<br />
</p><br />
<br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durrett and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
</div><br />
<br />
<h2 id="1.5">5. Our New Model</h2><br />
<p><br />
As mentioned before, the model proposed by Durrett and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it a <br />
biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive by <br />
outcompeting the other two strains, which will die. More specifically, we limited the resistance of the colicin-resistant <br />
bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and to the sensitive strain, <br />
and additionally the resistant strain would also be vulnerable to the colicin produced by the colicin-producer. <br />
Since which strain will be the one that survives is determined by very small differences in the initial concentrations of <br />
the three different populations of bacteria, in practice this systems becomes a randomizer because of the imprecisions in <br />
the measurements that result, for example, when using micropipettes. This randomizer describes a new competition dynamic <br />
that could not be reproduced in the previous model proposed by Durrett and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p>If we set the parameters as follows</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, we get a graph which clearly shows there are stable points on each of the three axes (Figure 1, Up).<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations we have set <br />
all of the three strains may ultimately survive for infinite peiriods of time. The differences between <br />
our model and the model of Durrett and Levin can be seen graphically in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail <br />
conditions of the model proposed by Durrett and Levin (indicated in black font)<br /><br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="1.6">6. The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its<br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in <br />
the production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h2 id="1.7">7. Making it Obvious</h2><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are <br />
paths that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this <br />
paths all have an approximately common origin. In this section we would like to show <br />
that the origin of these paths is practically the same, and that in that sense we have <br />
designed a true randomizer (since, as mentioned before, the imprecisions that result <br />
in the experimental measurements will make it impossible to make the initial<br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three different <br />
strains of <span class="name">E. coli</span> to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<br />
<p><br />
We modeled our results using Matlab. As can be seen in the graphs below, each of the strains <br />
can survive if their initial density in only tree hundredths (a.u.) greater than <br />
the other two strains' initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
</p><br />
<br/><br />
<br/><br />
<b>Reference </b><br /><br />
<p>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171.</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Modeling/RPS-game/RPS-gameTeam:Tokyo Tech/Modeling/RPS-game/RPS-game2011-10-28T10:50:41Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#1.1">1. Introduction</a></li><br />
<li><a href="#1.2">2. Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#1.3">3. How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#1.4">4. The Old Model</a></li><br />
<li><a href="#1.5">5. Our New Model</a></li><br />
<li><a href="#1.6">6. The Biological Meaning of our Model</a></li><br />
<li><a href="#1.7">7. Making it Obvious</a></li><br />
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<h1 id="1"> Survival of One Strain</h1><br />
<br />
<p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" width="500px" /><br />
</div><br />
<br />
<h2 id="1.1">1. Introduction: Minimal differences determine who will survive</h2><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a <br />
randomizer that can be used in our Rock-Paper-Scissors game, due to the fact that only one <br />
of the rival strains will survive. More specifically, we assign to each of the three <br />
rival strains either of Rock, Paper or Scissors, make them compete for survival and take <br />
the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h2 id="1.2">2. Adjusting the Model to create a True Randomizer</h2><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in 1996 by Durrett and Levin. <br />
In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. However, <br />
the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive (although it <br />
can dominate the system, i.e. have the highest population density, for definite periods <br />
of time). We will discuss more on the limitations we found in this model to be adopted as <br />
a randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h2 id="1.3">3. How the Three Types of Bacteria Compete for Survival</h2><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to <br />
outcompete their rivals: the production of a toxin (a bacteriocin called colicin)<br />
that is toxic to other strains, resistance to the toxin produced by other strains, <br />
and a higher birth rate than their rival strains. Namely, the three types <br />
of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) and colicin-sensitive <br />
<span class="name">E. coli</span> (S). The colicin-producer outcompetes the <br />
colicin-sensitive by producing the colicin. The colicin-sensitive bacteria <br />
outcompetes the colicin-resistant because its birth rate is higher <br />
than that of the colicin-resistant. The colicin-resistant outcompetes the <br />
colicin producer because its birth rate is higher than that of the colicin producer. <br />
The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" width="500px" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<br />
<h2 id="1.4">4. The Old Model</h2><br />
<br />
<p><br />
In the model described by Durrett and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). However, as will be explained afterwards, this results in a loss of balance that does not allow building a true randomizing system.<br />
</p><br />
<br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durrett and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
</div><br />
<br />
<h2 id="1.5">5. Our New Model</h2><br />
<p><br />
As mentioned before, the model proposed by Durrett and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it a <br />
biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive by <br />
outcompeting the other two strains, which will die. More specifically, we limited the resistance of the colicin-resistant <br />
bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and to the sensitive strain, <br />
and additionally the resistant strain would also be vulnerable to the colicin produced by the colicin-producer. <br />
Since which strain will be the one that survives is determined by very small differences in the initial concentrations of <br />
the three different populations of bacteria, in practice this systems becomes a randomizer because of the imprecisions in <br />
the measurements that result, for example, when using micropipettes. This randomizer describes a new competition dynamic <br />
that could not be reproduced in the previous model proposed by Durrett and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p>If we set the parameters as follows</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, we get a graph which clearly shows there are stable points on each of the three axes (Figure 1, Up).<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations we have set <br />
all of the three strains may ultimately survive for infinite peiriods of time. The differences between <br />
our model and the model of Durrett and Levin can be seen graphically in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail <br />
conditions of the model proposed by Durrett and Levin (indicated in black font)<br /><br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="1.6">6. The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its<br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in <br />
the production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h2 id="1.7">7. Making it Obvious</h2><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are <br />
paths that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this <br />
paths all have an approximately common origin. In this section we would like to show <br />
that the origin of these paths is practically the same, and that in that sense we have <br />
designed a true randomizer (since, as mentioned before, the imprecisions that result <br />
in the experimental measurements will make it impossible to make the initial<br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three different <br />
strains of <span class="name">E. coli</span> to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<br />
<p><br />
We modeled our results using Matlab. As can be seen in the graphs below, each of the strains <br />
can survive if their initial density in only tree hundredths (a.u.) greater than <br />
the other two strains' initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
</p><br />
<br/><br />
<br/><br />
<b>Reference </b><br /><br />
<p>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171.</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Modeling/RPS-game/RPS-gameTeam:Tokyo Tech/Modeling/RPS-game/RPS-game2011-10-28T10:49:49Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#1.1">1. Introduction</a></li><br />
<li><a href="#1.2">2. Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#1.3">3. How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#1.4">4. The Old Model</a></li><br />
<li><a href="#1.5">5. Our New Model</a></li><br />
<li><a href="#1.6">6. The Biological Meaning of our Model</a></li><br />
<li><a href="#1.7">7. Making it Obvious</a></li><br />
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<h1 id="1"> Survival of One Strain</h1><br />
<br />
<p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" width="500px" /><br />
</div><br />
<br />
<h2 id="1.1">1. Introduction: Minimal differences determine who will survive</h2><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a <br />
randomizer that can be used in our Rock-Paper-Scissors game, due to the fact that only one <br />
of the rival strains will survive. More specifically, we assign to each of the three <br />
rival strains either of Rock, Paper or Scissors, make them compete for survival and take <br />
the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h2 id="1.2">2. Adjusting the Model to create a True Randomizer</h2><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in 1996 by Durrett and Levin. <br />
In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. However, <br />
the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive (although it <br />
can dominate the system, i.e. have the highest population density, for definite periods <br />
of time). We will discuss more on the limitations we found in this model to be adopted as <br />
a randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h2 id="1.3">3. How the Three Types of Bacteria Compete for Survival</h2><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to <br />
outcompete their rivals: the production of a toxin (a bacteriocin called colicin)<br />
that is toxic to other strains, resistance to the toxin produced by other strains, <br />
and a higher birth rate than their rival strains. Namely, the three types <br />
of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) and colicin-sensitive <br />
<span class="name">E. coli</span> (S). The colicin-producer outcompetes the <br />
colicin-sensitive by producing the colicin. The colicin-sensitive bacteria <br />
outcompetes the colicin-resistant because its birth rate is higher <br />
than that of the colicin-resistant. The colicin-resistant outcompetes the <br />
colicin producer because its birth rate is higher than that of the colicin producer. <br />
The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<br />
<h2 id="1.4">4. The Old Model</h2><br />
<br />
<p><br />
In the model described by Durrett and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). However, as will be explained afterwards, this results in a loss of balance that does not allow building a true randomizing system.<br />
</p><br />
<br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durrett and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
</div><br />
<br />
<h2 id="1.5">5. Our New Model</h2><br />
<p><br />
As mentioned before, the model proposed by Durrett and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it a <br />
biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive by <br />
outcompeting the other two strains, which will die. More specifically, we limited the resistance of the colicin-resistant <br />
bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and to the sensitive strain, <br />
and additionally the resistant strain would also be vulnerable to the colicin produced by the colicin-producer. <br />
Since which strain will be the one that survives is determined by very small differences in the initial concentrations of <br />
the three different populations of bacteria, in practice this systems becomes a randomizer because of the imprecisions in <br />
the measurements that result, for example, when using micropipettes. This randomizer describes a new competition dynamic <br />
that could not be reproduced in the previous model proposed by Durrett and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p>If we set the parameters as follows</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, we get a graph which clearly shows there are stable points on each of the three axes (Figure 1, Up).<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations we have set <br />
all of the three strains may ultimately survive for infinite peiriods of time. The differences between <br />
our model and the model of Durrett and Levin can be seen graphically in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail <br />
conditions of the model proposed by Durrett and Levin (indicated in black font)<br /><br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="1.6">6. The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its<br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in <br />
the production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h2 id="1.7">7. Making it Obvious</h2><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are <br />
paths that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this <br />
paths all have an approximately common origin. In this section we would like to show <br />
that the origin of these paths is practically the same, and that in that sense we have <br />
designed a true randomizer (since, as mentioned before, the imprecisions that result <br />
in the experimental measurements will make it impossible to make the initial<br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three different <br />
strains of <span class="name">E. coli</span> to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<br />
<p><br />
We modeled our results using Matlab. As can be seen in the graphs below, each of the strains <br />
can survive if their initial density in only tree hundredths (a.u.) greater than <br />
the other two strains' initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
</p><br />
<br/><br />
<br/><br />
<b>Reference </b><br /><br />
<p>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171.</p><br />
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<ul><br />
<li><a href="#system">1. How our system works</a></li><br />
<li><a href="#new_parts">2. Data For Our Favorite New Parts</a></li><br />
<li><a href="#improve">3. Data For Pre-existing Parts</a></li><br />
<li><a href="#chared">4. We’ve Also Characterized the Following Parts</a></li><br />
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<h1> Data page </h1><br />
<br />
<p><br />
This page shows a list of all the parts that <br />
we have made or used in the project. <br />
Click on the link for each part to see <br />
more details about that part on the <br />
Registry of Standard Biological Parts. <br />
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<h2 id="system">1. How our system Works</h2><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/2/20/Allinone1.png" alt="allinone" width="800px" /><br />
<br />
<h2 id="new_parts">2. Data For Our Favorite New Parts</h2><br />
<br />
<ul><br />
<li> <br />
<a href="http://partsregistry.org/Part:BBa_K649001">BBa_K649001</a><br />
-- GFP regulated by 3OC12-HSL and LasR, BBa_K649001:<br /><br />
in the presence of 3OC12-HSL, lasI promoter can be induced to express a marker gene(<span class="gene">gfp</span>).<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649202">BBa_K649202</a><br />
-- PlacIQ-<i>lox71</i>-rbs-<span class="gene">rfp</span>-<i>lox66</i>-rbs-<span class="gene">gfp</span>, BBa_K649202:<br /><br />
in the presence of Cre, a marker gene (<span class="gene">rfp</span>) between <i>lox71</i> and <i>lox66</i> is knockout, and a marker gene (<span class="gene">gfp</span>) is expressed.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a><br />
-- Ptrc-rbs-<span class="gene">rocF</span>, BBa_K649301:<br /><br />
because arginase is constitutively expressed, the expression level of urea in <span class="name">E. coli</span> transformed with BBa_K649301 was higher than geneless <span class="name">E. coli</span>.<br />
</li><br />
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<br />
<h2 id="improve">3. Data For Pre-existing Parts</h2><br />
<br />
<ul><br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_J64010:Experience">BBa_J64010:Experience</a><br />
-- lasI promoter, BBa_J64010 (Voigt Lab, 2007):<br /><br />
fluorescence intensity of PlasI (BBa_J64010) -<span class="gene">gfp</span> did not change before and after 3OC12-HSL induction.<br />
</li> <br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_I751101:Experience">BBa_I751101:Experience</a><br />
-- J540140 dPr + hybrid promoter (Plux-lac), BBa_I751101 (Tokyo Tech, 2007):<br /><br />
fluorescence intensity of BBa_I751101 was increased by both 3OC6-HSL induction and IPTG induction.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K117002:Experience">BBa_K117002:Experience</a><br />
-- LsrA promoter (indirectly activated by AI-2), BBa_K117002 (NTU-Singapore, 2008):<br /><br />
in the absence of LsrR, fluorescence intensity of PlsrA (BBa_K117002) -<span class="gene">gfp</span> was lower than that of promoterless negative control.<br />
</li><br />
</ul><br />
<br />
<h2 id="chared">4. We’ve Also Characterized the Following Parts</h2><br />
<br />
<ul><br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a><br />
-- PlsrA-rbs-<span class="gene">gfp</span>, BBa_K649104:<br /><br />
in the absence of LsrR, fluorescence intensity of BBa_K649104 was much higher than promoterless-<span class="gene">gfp</span> (negative control).<br />
</li><br />
<br />
<li> <br />
<a href="http://partsregistry.org/Part:BBa_K649105">BBa_K649105</a><br />
-- PlsrA-rbs-<span class="gene">gfp</span>-TT-PlsrR-rbs-<span class="gene">lsrR</span>, BBa_K649105:<br /><br />
PlsrA (BBa_K649100) was repressed by LsrR and fluorescence intensity of BBa-K649105 decreased 3-fold.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649200">BBa_K649200</a><br />
-- PlacIQ-<i>lox2272</i>-rbs-<span class="gene">gfp</span>-<i>lox2272</i>, BBa_K649200:<br /><br />
in the presence of Cre, the sequence between two <i>lox2272</i> is knockout.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649201">BBa_K649201</a><br />
-- PlacIQ-<i>lox2272</i>-rbs-<span class="gene">rfp</span>-<i>lox2272</i>-rbs-<span class="gene">gfp</span>, BBa_K649201:<br /><br />
in the presence of Cre, a marker gene (<span class="gene">rfp</span>) between two <i>lox2272</i> is knockout, and a marker gene (<span class="gene">gfp</span>) is expressed.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649303">BBa_K649303</a><br />
-- PlacIQ-rbs-<i>ispS</i>, BBa_K649303:<br /><br />
we confirmed that E. coli introduced <i>ispS</i> produced isoprene, by means of using electron-ionization Gas Chromatography-Mass Spectrometry equipment.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649401">BBa_K649401</a><br />
-- Arg box, BBa_K649401:<br /><br />
because Arg Box is the arginine operator which the arginine repressor can bind to, the expression level of urea in <span class="name">E. coli</span> transformed with BBa_K649401 was higher than mock <span class="name">E. coli</span>.<br />
</li><br />
<br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649402">BBa_K649402</a><br />
-- Ptrc-rbs-<span class="gene">rocF</span>-Arg box, BBa_K649402:<br /><br />
because we transformed <span class="name">E. coli</span> with BBa_K649402 on low copy plasmid, Arg box did not replicated adequately and we could not confirm whether Arg box was working or not.<br />
</li><br />
<br />
</ul><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/DataPage.htmTeam:Tokyo Tech/DataPage.htm2011-10-28T10:48:46Z<p>Takuya 1613: </p>
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<h1> Data page </h1><br />
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<h2 id="system">1. How our system Works</h2><br />
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<li> <br />
<a href="http://partsregistry.org/Part:BBa_K649001">BBa_K649001</a><br />
-- GFP regulated by 3OC12-HSL and LasR, BBa_K649001:<br /><br />
in the presence of 3OC12-HSL, lasI promoter can be induced to express a marker gene(<span class="gene">gfp</span>).<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649202">BBa_K649202</a><br />
-- PlacIQ-<i>lox71</i>-rbs-<span class="gene">rfp</span>-<i>lox66</i>-rbs-<span class="gene">gfp</span>, BBa_K649202:<br /><br />
in the presence of Cre, a marker gene (<span class="gene">rfp</span>) between <i>lox71</i> and <i>lox66</i> is knockout, and a marker gene (<span class="gene">gfp</span>) is expressed.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a><br />
-- Ptrc-rbs-<span class="gene">rocF</span>, BBa_K649301:<br /><br />
because arginase is constitutively expressed, the expression level of urea in <span class="name">E. coli</span> transformed with BBa_K649301 was higher than geneless <span class="name">E. coli</span>.<br />
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<h2 id="improve">3. Data For Pre-existing Parts</h2><br />
<br />
<ul><br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_J64010:Experience">BBa_J64010:Experience</a><br />
-- lasI promoter, BBa_J64010 (Voigt Lab, 2007):<br /><br />
fluorescence intensity of PlasI (BBa_J64010) -<span class="gene">gfp</span> did not change before and after 3OC12-HSL induction.<br />
</li> <br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_I751101:Experience">BBa_I751101:Experience</a><br />
-- J540140 dPr + hybrid promoter (Plux-lac), BBa_I751101 (Tokyo Tech, 2007):<br /><br />
fluorescence intensity of BBa_I751101 was increased by both 3OC6-HSL induction and IPTG induction.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K117002:Experience">BBa_K117002:Experience</a><br />
-- LsrA promoter (indirectly activated by AI-2), BBa_K117002 (NTU-Singapore, 2008):<br /><br />
in the absence of LsrR, fluorescence intensity of PlsrA (BBa_K117002) -<span class="gene">gfp</span> was lower than that of promoterless negative control.<br />
</li><br />
</ul><br />
<br />
<h2 id="chared">4. We’ve Also Characterized the Following Parts</h2><br />
<br />
<ul><br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a><br />
-- PlsrA-rbs-<span class="gene">gfp</span>, BBa_K649104:<br /><br />
in the absence of LsrR, fluorescence intensity of BBa_K649104 was much higher than promoterless-<span class="gene">gfp</span> (negative control).<br />
</li><br />
<br />
<li> <br />
<a href="http://partsregistry.org/Part:BBa_K649105">BBa_K649105</a><br />
-- PlsrA-rbs-<span class="gene">gfp</span>-TT-PlsrR-rbs-<span class="gene">lsrR</span>, BBa_K649105:<br /><br />
PlsrA (BBa_K649100) was repressed by LsrR and fluorescence intensity of BBa-K649105 decreased 3-fold.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649200">BBa_K649200</a><br />
-- PlacIQ-<i>lox2272</i>-rbs-<span class="gene">gfp</span>-<i>lox2272</i>, BBa_K649200:<br /><br />
in the presence of Cre, the sequence between two <i>lox2272</i> is knockout.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649201">BBa_K649201</a><br />
-- PlacIQ-<i>lox2272</i>-rbs-<span class="gene">rfp</span>-<i>lox2272</i>-rbs-<span class="gene">gfp</span>, BBa_K649201:<br /><br />
in the presence of Cre, a marker gene (<span class="gene">rfp</span>) between two <i>lox2272</i> is knockout, and a marker gene (<span class="gene">gfp</span>) is expressed.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649303">BBa_K649303</a><br />
-- PlacIQ-rbs-<i>ispS</i>, BBa_K649303:<br /><br />
we confirmed that E. coli introduced ispS produced isoprene, by means of using electron-ionization Gas Chromatography-Mass Spectrometry equipment.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649401">BBa_K649401</a><br />
-- Arg box, BBa_K649401:<br /><br />
because Arg Box is the arginine operator which the arginine repressor can bind to, the expression level of urea in <span class="name">E. coli</span> transformed with BBa_K649401 was higher than mock <span class="name">E. coli</span>.<br />
</li><br />
<br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649402">BBa_K649402</a><br />
-- Ptrc-rbs-<span class="gene">rocF</span>-Arg box, BBa_K649402:<br /><br />
because we transformed <span class="name">E. coli</span> with BBa_K649402 on low copy plasmid, Arg box did not replicated adequately and we could not confirm whether Arg box was working or not.<br />
</li><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/DataPage.htmTeam:Tokyo Tech/DataPage.htm2011-10-28T10:41:48Z<p>Takuya 1613: </p>
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<li><a href="#chared">4. We’ve Also Characterized the Following Parts</a></li><br />
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<h1> Data page </h1><br />
<br />
<p><br />
This page shows a list of all the parts that <br />
we have made or used in the project. <br />
Click on the link for each part to see <br />
more details about that part on the <br />
Registry of Standard Biological Parts. <br />
For a brief overview of our main results, <br />
please have a look at our Main Results page.<br />
</p><br />
<br />
<h2 id="system">1. How our system Works</h2><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/2/20/Allinone1.png" alt="allinone" width="800px" /><br />
<br />
<h2 id="new_parts">2. Data For Our Favorite New Parts</h2><br />
<br />
<ul><br />
<li> <br />
<a href="http://partsregistry.org/Part:BBa_K649001">BBa_K649001</a><br />
-- GFP regulated by 3OC12-HSL and LasR, BBa_K649001:<br /><br />
in the presence of 3OC12-HSL, lasI promoter can be induced to express a marker gene(<span class="gene">gfp</span>).<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649202">BBa_K649202</a><br />
-- PlacIQ-<i>lox71</i>-rbs-<span class="gene">rfp</span>-<i>lox66</i>-rbs-<span class="gene">gfp</span>, BBa_K649202:<br /><br />
in the presence of Cre, a marker gene (<span class="gene">rfp</span>) between <i>lox71</i> and <i>lox66</i> is knockout, and a marker gene (<span class="gene">gfp</span>) is expressed.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a><br />
-- Ptrc-rbs-<span class="gene">rocF</span>, BBa_K649301:<br /><br />
because arginase is constitutively expressed, the expression level of urea in <span class="name">E. coli</span> transformed with BBa_K649301 was higher than geneless <span class="name">E. coli</span>.<br />
</li><br />
</ul><br />
<br />
<h2 id="improve">3. Data For Pre-existing Parts</h2><br />
<br />
<ul><br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_J64010:Experience">BBa_J64010:Experience</a><br />
-- lasI promoter, BBa_J64010 (Voigt Lab, 2007):<br /><br />
fluorescence intensity of PlasI (BBa_J64010) -<span class="gene">gfp</span> did not change before and after 3OC12-HSL induction.<br />
</li> <br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_I751101:Experience">BBa_I751101:Experience</a><br />
-- J540140 dPr + hybrid promoter (Plux-lac), BBa_I751101 (Tokyo Tech, 2007):<br /><br />
fluorescence intensity of BBa_I751101 was increased by both 3OC6-HSL induction and IPTG induction.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K117002:Experience">BBa_K117002:Experience</a><br />
-- LsrA promoter (indirectly activated by AI-2), BBa_K117002 (NTU-Singapore, 2008):<br /><br />
in the absence of LsrR, fluorescence intensity of PlsrA (BBa_K117002) -<span class="gene">gfp</span> was lower than that of promoterless negative control.<br />
</li><br />
</ul><br />
<br />
<h2 id="chared">4. We’ve Also Characterized the Following Parts</h2><br />
<br />
<ul><br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a><br />
-- PlsrA-rbs-<span class="gene">gfp</span>, BBa_K649104:<br /><br />
in the absence of LsrR, fluorescence intensity of BBa_K649104 was much higher than promoterless-<span class="gene">gfp</span> (negative control).<br />
</li><br />
<br />
<li> <br />
<a href="http://partsregistry.org/Part:BBa_K649105">BBa_K649105</a><br />
-- PlsrA-rbs-<span class="gene">gfp</span>-TT-PlsrR-rbs-<span class="gene">lsrR</span>, BBa_K649105:<br /><br />
PlsrA (BBa_K649100) was repressed by LsrR and fluorescence intensity of BBa-K649105 decreased 3-fold.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649200">BBa_K649200</a><br />
-- PlacIQ-<i>lox2272</i>-rbs-<span class="gene">gfp</span>-<i>lox2272</i>, BBa_K649200:<br /><br />
in the presence of Cre, the sequence between two <i>lox2272</i> is knockout.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649201">BBa_K649201</a><br />
-- PlacIQ-<i>lox2272</i>-rbs-<span class="gene">rfp</span>-<i>lox2272</i>-rbs-<span class="gene">gfp</span>, BBa_K649201:<br /><br />
in the presence of Cre, a marker gene (<span class="gene">rfp</span>) between two <i>lox2272</i> is knockout, and a marker gene (<span class="gene">gfp</span>) is expressed.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649303">BBa_K649303</a><br />
-- PlacIQ-rbs-<i>ispS</i>, BBa_K649303:<br /><br />
<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649401">BBa_K649401</a><br />
-- Arg box, BBa_K649401:<br /><br />
because Arg Box is the arginine operator which the arginine repressor can bind to, the expression level of urea in <span class="name">E. coli</span> transformed with BBa_K649401 was higher than mock <span class="name">E. coli</span>.<br />
</li><br />
<br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649402">BBa_K649402</a><br />
-- Ptrc-rbs-<span class="gene">rocF</span>-Arg box, BBa_K649402:<br /><br />
because we transformed <span class="name">E. coli</span> with BBa_K649402 on low copy plasmid, Arg box did not replicated adequately and we could not confirm whether Arg box was working or not.<br />
</li><br />
<br />
</ul><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/DataPage.htmTeam:Tokyo Tech/DataPage.htm2011-10-28T10:31:22Z<p>Takuya 1613: </p>
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<h1> Data page </h1><br />
<br />
<p><br />
This page shows a list of all the parts that <br />
we have made or used in the project. <br />
Click on the link for each part to see <br />
more details about that part on the <br />
Registry of Standard Biological Parts. <br />
For a brief overview of our main results, <br />
please have a look at our Main Results page.<br />
</p><br />
<br />
<h2 id="system">1. How our system Works</h2><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/2/20/Allinone1.png" alt="allinone" width="800px" /><br />
<br />
<h2 id="new_parts">2. Data For Our Favorite New Parts</h2><br />
<br />
<ul><br />
<li> <br />
<a href="http://partsregistry.org/Part:BBa_K649001">BBa_K649001</a><br />
-- GFP regulated by 3OC12-HSL and LasR, BBa_K649001:<br /><br />
in the presence of 3OC12-HSL, lasI promoter can be induced to express a marker gene(<span class="gene">gfp</span>).<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649202">BBa_K649202</a><br />
-- PlacIQ-lox71-rbs-<span class="gene">rfp</span>-lox66-rbs-<span class="gene">gfp</span>, BBa_K649202:<br /><br />
in the presence of Cre, a marker gene (<span class="gene">rfp</span>) between lox71 and lox66 is knockout, and a marker gene (<span class="gene">gfp</span>) is expressed.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a><br />
-- Ptrc-rbs-<span class="gene">rocF</span>, BBa_K649301:<br /><br />
because arginase is constitutively expressed, the expression level of urea in <span class="name">E. coli</span> transformed with BBa_K649301 was higher than geneless <span class="name">E. coli</span>.<br />
</li><br />
</ul><br />
<br />
<h2 id="improve">3. Data For Pre-existing Parts</h2><br />
<br />
<ul><br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_J64010:Experience">BBa_J64010:Experience</a><br />
-- lasI promoter, BBa_J64010 (Voigt Lab, 2007):<br /><br />
fluorescence intensity of PlasI (BBa_J64010) -<span class="gene">gfp</span> did not change before and after 3OC12-HSL induction.<br />
</li> <br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_I751101:Experience">BBa_I751101:Experience</a><br />
-- J540140 dPr + hybrid promoter (Plux-lac), BBa_I751101 (Tokyo Tech, 2007):<br /><br />
fluorescence intensity of BBa_I751101 was increased by both 3OC6-HSL induction and IPTG induction.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K117002:Experience">BBa_K117002:Experience</a><br />
-- LsrA promoter (indirectly activated by AI-2), BBa_K117002 (NTU-Singapore, 2008):<br /><br />
in the absence of LsrR, fluorescence intensity of PlsrA (BBa_K117002) -<span class="gene">gfp</span> was lower than that of promoterless negative control.<br />
</li><br />
</ul><br />
<br />
<h2 id="chared">4. We’ve Also Characterized the Following Parts</h2><br />
<br />
<ul><br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a><br />
-- PlsrA-rbs-<span class="gene">gfp</span>, BBa_K649104:<br /><br />
in the absence of LsrR, fluorescence intensity of BBa_K649104 was much higher than promoterless-<span class="gene">gfp</span> (negative control).<br />
</li><br />
<br />
<li> <br />
<a href="http://partsregistry.org/Part:BBa_K649105">BBa_K649105</a><br />
-- PlsrA-rbs-<span class="gene">gfp</span>-TT-PlsrR-rbs-<span class="gene">lsrR</span>, BBa_K649105:<br /><br />
PlsrA (BBa_K649100) was repressed by LsrR and fluorescence intensity of BBa-K649105 decreased 3-fold.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649200">BBa_K649200</a><br />
-- PlacIQ-lox2272-rbs-<span class="gene">gfp</span>-lox2272, BBa_K649200:<br /><br />
in the presence of Cre, the sequence between two lox2272 is knockout.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649201">BBa_K649201</a><br />
-- PlacIQ-lox2272-rbs-<span class="gene">rfp</span>-lox2272-rbs-<span class="gene">gfp</span>, BBa_K649201:<br /><br />
in the presence of Cre, a marker gene (<span class="gene">rfp</span>) between two lox2272 is knockout, and a marker gene (<span class="gene">gfp</span>) is expressed.<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649303">BBa_K649303</a><br />
-- PlacIQ-rbs-ispS, BBa_K649303:<br /><br />
<br />
</li><br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649401">BBa_K649401</a><br />
-- Arg box, BBa_K649401:<br /><br />
because Arg Box is the arginine operator which the arginine repressor can bind to, the expression level of urea in <span class="name">E. coli</span> transformed with BBa_K649401 was higher than mock <span class="name">E. coli</span>.<br />
</li><br />
<br />
<br />
<li><br />
<a href="http://partsregistry.org/Part:BBa_K649402">BBa_K649402</a><br />
-- Ptrc-rbs-<span class="gene">rocF</span>-Arg box, BBa_K649402:<br /><br />
because we transformed <span class="name">E. coli</span> with BBa_K649402 on low copy plasmid, Arg box did not replicated adequately and we could not confirm whether Arg box was working or not.<br />
</li><br />
<br />
</ul><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/index.htmTeam:Tokyo Tech/Projects/Urea-cooler/index.htm2011-10-28T10:30:25Z<p>Takuya 1613: </p>
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<ul><br />
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<a href="#abst">1. Abstruct</a><br />
</li><br />
<li><br />
<a href="#2.Gene">2. Genetic Engineering for Urea Production</a><br />
<ul> <br />
<li><a href="#2.1Intro">2.1 Introduction</a></li><br />
<li><a href="#2.2">2.2 Charcterization of rocF and Arg box</a></li><br />
<li><a href="#2.3">2.3 Characterization of Ptrc-RBS-rocF-Arg box</a></li><br />
<li><a href="#2.4">2.4 Use of mutation in argR gene</a></li><br />
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<a href="#Flux">3. Flux analysis for providing more urea </a><br />
<ul><br />
<li><a href="#abstract">3.1 Abstract</a></li><br />
<li><a href="#intro">3.2 Introduction</a></li><br />
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<h1> Urea cooler </h1><br />
<br />
<p><br />
<h2 id="abst">1. Abstract</h2><br />
<p><br />
Coolers can be made by dissolving urea in water because it is an endothermic reaction. So, in order to obtain urea, we made the urea cycle in<i> E.coli</i> by introducing <br />
<i>rocF</i> gene encoding arginase, which is an enzyme that converts L-arginine to L-ornithine and urea.<br />
But just by introducing <i>rocF</i>, <br />
only a little amount of urea can be produced because arginine biosynthesis is repressed by the arginine repressor. Therefore, we tried to derepress arginine biosynthesis in two ways. One is by introducing arginine operator sequences(Arg boxes), which bind the arginine repressor. The other is by using a strain that carries a mutation in a gene which encodes arginine repressor. <br />
Furthermore, we studied elementary flux modes to provide more urea. As a result, <br />
we found out that the artificial urea production system is robust in a stoichiometric point of view. The analysis also <br />
revealed that supplementation of arginine, glutamic acid and aspartic acid would increase urea production rate.<br /><br />
<table style="text-align:center;" align="center"><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/d/de/Characterization_rocF_and_Arg_box.png" alt="Assay data" width="350px" align="center" /></td><br />
<td><img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="350px" /></td><br />
</tr><br />
<tr><br />
<td>Fig.3 Urea concentration in growth media 1 hour after IPTG induction </td><br />
<td> Fig.5 The reactions related with the urea cycle</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<h2 id="2.Gene">2. Genetic Engineering for Urea Production</h2><br />
<h3 id="2.1Intro">2.1 Introduction</h3><br />
<p><br />
Coolers can be made by dissolving urea in water,<br />
since dissolution of urea in water is an endothermic reaction (-57.8 cal/g). So we came up with an idea of creating <i>E. coli</i> that synthesizes urea.<br />
Originally, <i>E. coli</i> has all the enzymes in the urea cycle except for arginase, which converts L-arginine into L-ornithine and urea(Fig.1). so we attempted to complete the urea cycle in <i>E. coli</i> and obtain urea. <br /><br />
<br />
In this work, introduction of the <i>Bacillus subtilis</i> <i>rocF</i> gene which encodes arginase on a <br />
standardized plasmid completed urea cycle and enabled<i> E.coli</i> to produce urea <br />
as reported by TUCHMAN <i>et al</i>.,(1997). <br /><br />
<br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/2/21/TokyoTech_urea-cycle1.png" alt="Urea cycle; Fig1" width="700px" align="center" /><br />
<div class="graph_title"><br />
Fig.1 Addition of a gene which encoding arginase completes urea cycle in <i>E.coli</i>.<br />
</div></center><br /><br />
<br />
<p><br />
However, just by introducing arginase, <i>E. coli</i> produces <br />
only a little amount of urea. TUCHMAN <i>et al</i>. proposed that catabolite <br />
repression in arginine biosynthesis pathway is the main reason for <br />
the low efficiency of production(TUCHMAN <i>et al</i>., 1997). The bacterial <br />
arginine biosynthetic genes are all regulated via a common repressor <br />
protein (arginine repressor) encoded by the <i>argR</i> gene. Arginine represor is activated in the presence of arginine (Fig.2).<br/><br />
TUCHMAN <i>et al</i>. circumvented the repression by introduction of <br />
arginine operator sequences (Arg boxes), which bind the arginine repressor. <br />
With the arginine repressor binding to Arg boxes, the amount of the arginine <br />
repressor which can repress arginine biosynthesis is reduced. <br />
In this work, we tried two ways of derepressing arginine biosynthesis. One way <br />
is introducing the Arg boxes as previous work. The other way is using an <br />
<i>E. coli</i> strain that has an <i>argR</i> deletion genotype so that <br />
the repressor is not synthetized. <br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/3/30/Arginine_repressor.png" alt="Arginine biosynthesis is repressed by arginine repressor in the presence of arginine." align="center" width="600px"/><br />
<div class="graph_title"><br />
Fig.2 Arginine biosynthesis is repressed by arginine repressor in the presence of arginine.<br />
</div><br />
</center><br />
<br />
<h3 id="2.2">2.2 Charcterization of <i>rocF</i> and Arg box</h3><br />
<br />
<p><br />
Introduction of <i>rocF</i> gene on our part <a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a> led to more urea production compared to negative controls. Therefore, we confirmed that insertion of <i>rocF</i> gene resulted in arginase production and completed the urea cycle in <span class="name">E. coli</span> as expected.<br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/d/de/Characterization_rocF_and_Arg_box.png" align="center" width="400px"/></center><br />
<div class="graph_title"><br />
Fig.3 Urea concentration in growth media when <i>rocF</i> or Arg box were introduced strain MG1655. </div></center><br /><br />
<br />
<p><br />
As a way to derepress arginine biosynthesis we introduced Arg box on our part <a href="http://partsregistry.org/Part:BBa_K649401">BBa_K649401</a> in addition to <i>rocF</i> gene (<a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a>) led to production of even more urea. These results show that Arg boxes are effectively derepressing arginine production by deactivating arginine repressor.<br />
</p><br />
<big><b>Materials and Methods</b></big><br /><br />
<p>Materials and methods and other detailed information about this study are shown <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#1.1">here</a>.</p><br />
<h3 id="2.3">2.3 Characterization of Ptrc-RBS-<i>rocF</i>-Arg box</h3><br />
<p><br />
In this study Arg boxes and <i>rocF</i> were introduced separately on high-copy-number plasmids (pSB1C3) and low-copy-number plasmids (pSB3K3). We also tested the effect of Arg boxes when they were introduced downstream of <i>rocF</i> gene on low-copy-number plasmids (pSB3K3 and pSB6A1). Here, Arg boxes didn’t work effectively. This is probably because a low-copy-number plasmid is not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. Detailed information about this study is shown <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#2.1">here</a>.<br />
</p><br />
<br />
<h3 id="2.4">2.4 Use of mutation in <span class="gene">argR</span> gene</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/0/08/RocF_on_pSB3K3.png" align="center" width="400px"/><br />
<div class="graph_title"><br />
Fig.4 Urea concentration in growth media when <i>rocF</i> gene on pSB3K3 was introduced in strain MG1655 (<span class="gene">argR</span> +) or JE6852 (<span class="gene">argR</span> -).<br />
</div><br />
</center><br /><br />
<p><br />
As another way to derepress arginine biosynthesis, we used an <span class="name">E. coli</span> strain that has an <span class="gene">argR</span> deletion genotype (JE6852, This strain was obtained from National Institute of Genetics). In our assay results, urea production in JE6852 is much more than in MG1655 as expected.<br />
</p><br />
<big><b>Methods</b></big><br /><br />
<p><br />
Materials and methods are described <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#2.1">here</a>.<br />
</p><br />
<br />
<h3 id="Flux"> Flux analysis for providing more urea </h3><br />
<h4 id="abstract">3.1 Abstract </h4><br />
<p>This section is about a metabolic engineering study we did about the urea cycle. On the first part we show how we used &ldquo;elementary flux modes&rdquo; (Schuster <i>et al</i>., 2000) to analyze the function of the compounds involved in the urea cycle. Mainly we deduced which compounds act as sources of carbon and sources of nitrogen for the production of urea. On the second part of this study we show how we determined elementary flux modes of the urea cycle to find ways to increase the yield of urea. We focused on a strategy which involves increasing the concentration of four components of the cycle and which we concluded would yield more urea. To confirm our results future experiments will be done.</p><br />
<br />
<div class="graph_title"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="750px" /><br />
<br />Fig.5 The reactions related with the urea cycle<br />
</div><br /><br />
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene"><i>rocF</i></span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.<br /><br />
<br />
<br />
<h5 id="intro">3.2 Introduction</h5><br />
<big><b>3.2.1 What is Elementary Flux Modes?</b></big><br /><br />
<p>In Metabolic Engineering, mathematical modeling is an effective way to increase the products of a reaction. In particular, Flux Analysis, which is based on the hypothesis that the system is in a steady state, is effective to find how to increase these products. The concept of elementary flux mode provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. As a method of metabolic flux analysis, it is based on the hypothesis that the concentration of the reactants and products involved in the cycle does not change. </p><br /><br />
<br />
<big><b>3.2.2 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</b></big><br /><br />
<p>By determining the elementary flux modes of a cycle we can have a more clear view of the function of each of the compounds involved in the cycle being analyzed. Based on the elementary flux modes of the urea cycle, in this study we could deduce that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production and that both L-glutamine and NH<sub>3</sub> act as nitrogen sources for the formation of urea. </p><br /><br />
<cener><br />
<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="200px" /><br />
<span style="font-size:12px;">&rarr;</span><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="200px" /><br />
<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="Fig7" width="400px" /><br />
</center><br /><br />
<br />
<big><b>3.2.3 Finding Modes to Increase the Urea production by <i>E. coli</i></b></big><br />
<p>In this study we determined elementary flux modes to maximize urea production by <i>E. coli</i>. We found that there are two main strategies to increase urea production: one is to increase the amount of carbamoyl phosphate (which formation is known to be the rate-limiting step of the urea cycle). The other one is to increase the concentration of four components of the urea cycle: L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine. We deduced the latter strategy by determining the elementary modes of the urea cycle, and therefore in this study we will focus on the description of this strategy.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="500px" /><br /><br />
Fig.8 Two ways to increase urea production <br /><br /><br />
<br />
<h6 id=results>3.3 Results</h6><br />
<br />
<p>In our study, we considered the enzymatic reactions shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a> to determine the elementary flux modes related to urea production by <i>E. coli</i>. The scheme the overall reaction system is shown in Fig.5 below.</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="600px" /></div><br />
<div class="graph_title"><br />
<br />Fig.5 The reactions related with the urea cycle<br />
</div><br />
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene">rocF</span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.<br /><br />
<br /><br />
<p>By determining the elementary flux modes to produce urea inside <i>E. coli</i>, we found two important results: </p><br />
<p>1. We confirmed both L-glutamine and NH<sub>3</sub> act as nitrogen providers in the urea cycle, as well as deducing that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production. These modes did not make use of organic intermediates. Even though L-glutamine is consumed in order to to transfer the side-chain ammonium group needed for the production of carbamoyl phosphate (which in turn transfers the ammonium group to the urea cycle), free ammonium ion can restore L-glutamine from L-glutamate (which is a byproduct of the reaction that yields carbamoyl phosphate as a product).<br />
</p><br />
<p>2. We concluded that increasing the concentration of L-ornithine will increase the concentration of three related compounds (L-citrulline, N-(L-arginino)succinate, and L-arginine) and this will ultimately lead to an increase in the production of urea. We also noted that since the L-aspartate amino acid, which is needed in the urea cycle we considered(Fig. 5), is normally consumed in protein biosynthesis, so it should be supplied in the culture medium or synthetized by <span class="name">E. coli</span> in order to be able to increase the amount of urea and to maintain the cycles that produce it. </p><br />
<br />
<p>Below is a detailed description of these three results.</p><br />
<big><b>3.3.1 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</b></big><br /><br />
<p>The first step was to determine the flux modes which need of L-glutamine as an input (Mendel <i>et al</i>., 1996). We did this by calculations based on a matrix as the tableau shown below.</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="800px" /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="800px" /><br />
</center><br />
<br /><a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/method">Details about the calculations can be found here</a><br />
<br />
<p>We found eight modes that can produce urea without using organic intermediates. These are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem1">Fig.6</a>. Each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table4">Table 4</a>. In particular, we focused on one the mode displayed in Fig.7.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="fig7" width="750px" /></div><br />
<div class="graph_title"><br />
<div style="font-size:larger"><br />
<b>2NH<sub>3</sub> + HCO<sub>3</sub><sup>-</sup> + 3ATP + H<sub>2</sub>O + NADPH + NAD<sup>+</sup> <br />
→ Urea + 2ADP + AMP + 2Pi + PPi + NADP<sup>+</sup> + NADH</b><br />
</div><br />
Fig.7 One of the urea producing cycles leaded by the concept of elementary flux modes<br />
*The numbers indicate the relative flux carried by the enzymes.<br /><br /><br />
</div><br />
As shown in Fig.7, we deduced that the carbon atom of urea is provided from HCO<sub>3</sub><sup>-</sup> , which is a byproduct of respiration and therefore is already an abundant compound in the bacterial cytoplasm. On the other hand, we also confirmed that carbamoyl phosphate is a nitrogen source for urea production.We also found that the function of L-glutamine in the urea cycle is to provide nitrogen for urea production via carbamoyl phosphate, because ammonium ion can restore L-glutamine from L-glutamate (which is a<br />
byproduct of the reaction that yields carbamoyl phosphate as a product).This conclusion was confirmed experimentally by Mendel <i>et al</i>. (1996). Also, since only providing a nitrogen source is enough to increase urea production by <i>E. coli</i>, we can also conclude that the aritificial urea cycle in <i>E. coli</i> is stoichiometrically well designed. By comparing Fig.5 and Fig.7 we can also observe that, in Fig.7, the reaction which converts L-glutamate to L-ornithine is not needed for urea production. </p><br />
<br />
<big><b>3.3.2. Finding Modes to Increase the Urea production by <i>E. coli</i></b></big><br /><br />
<p>There are two ways to obtain more products from a cycle of reactions: increasing the speed the reactions and increasing the concentration of the reactants. This becomes obvious if we think of the cycle as a track which is travelled by cars (the reactants), and the products as the total sum of the number of laps made by every car. If we double the speed of the cars the number of laps will also double (Fig. 8, lower left). Similarly, if we double the number of cars the number of laps will double as well (Fig. 8, lower right). We applied this analogy to the urea cycle, where the metabolites in the cycle are represented by the cars and the total number of laps represents the total urea yield (as shown in the figure below).<br /><br />
Increasing the velocity of the cars corresponds to increasing the amount of carbamoyl phosphate in the urea cycle, because the reaction which converts L-glutamine to carbamoyl phosphate is the rate-limiting reaction of the cycle. On the other hand, increasing the number of the cars correspond to increasing the concentration of the compounds of the urea cycle. We focused on increasing the concentration the compounds of the urea cycle to find ways to increase the urea yield.<br /><br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="700px" /><br /><br />
Fig.8 Two ways to increase urea production <br /><br /><br />
L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine are four important compounds of the urea cycle. As can be seen I Fig.7, these compounds form a sub-cycle that directly yields urea. Therefore, by increasing the yield of this cycle we can increase the production of urea in <i>E. coli</i>.</p><br />
<br />
<p>We determined the elementary modes which produce these four important compounds. <br />
All elementary flux modes which produce these compounds from L-glutamine or from compounds in TCA cycle produce L-ornithine as intermediate or final product (these modes are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem2">Fig.9</a> and each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table5">Table 5</a>, it can be concluded that increasing the concentration of L-ornithine will increase the production of urea. One of the L-ornithine producing modes is shown in Fig.10.<br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/8/89/Urea-fig11.png" alt="fig11" width="600px" /><br />
</center><br />
<div class="graph_title"><br />
<div style="font-size:larger"><br />
<small><b>2-oxoglutarate + NH<sub>3</sub> + acetyl-CoA + ATP + 3NADPH + 3H<sup>+</sup> <br />
→ L-ornithine + CoASH + acetate + ADP + Pi + H<sub>2</sub>O + 3NADP<sup>+</sup></b></small><br />
</div><br />
Fig.10 One of the L-ornithine producing pathways from intermediates of TCA cycle<br /><br />
*The numbers indicate the relative flux carried by the enzymes.<br /><br /><br />
</div><br />
<br />
<p>The reactions we determined increase the above mentioned four compounds of the urea cycle are shown in Fig. 9. All modes include the reaction that yields L-ornithine by converting L-glutamate to L-ornithine. <br /><br />
We also confirmed that <span class="name">E. coli</span> has no feasible routes for production of these four components other than those indicated in Fig.5. Therefore, we can conclude that the reaction which converts L-glutamate to L-ornithine is a key reaction to increase the reaction rates in the urea cycle and thereby to increase urea production. It should be noted that one of the reactions of the cycle shown in Fig. 5 (the one in the lowest part of the image) requires ATP, NADPH, Acetyl-CoA, and L-glutamate. With the exception of L-glutamate, all of these compounds are already abundant in the cell. Therefore, in future wet experiments, we will focus on studying the effects of supplying L-glutamate to <i>E. coli</i>. We will confirm that by supplying L-glutamate the concentration of intermediates like L-ornithine can be increased and therefore urea production can be increased.<br /><br />
</p><br />
<p>Furthermore, to supply L-glutamine, L-glutamate and L-arginine is effective way to increase the amount of ornithine.(Fig.11)</p><br />
<div arign="center"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a6/Urea_modeling_overview.png" alt="fig.11a" width="600px" /><br /><br />
<span class="graph_title">Fig.11 Ornithine is made from L-glutamine, L-glutamate and L-arginine</span><br />
</div><br />
<p><br />
We also noted that since L-aspartate is consumed in protein biosynthesis, this amino acid should be supplied from in the medium or produced by <i>E. coli</i> itself not only for increasing the amount of urea production, but also for maintaining the cycle.<br /></p><br />
<br />
<p>In conclusion, increasing the concentration of L-glutamine, L-glutamate, L-arginine and L-aspartate is an effective way to increase the amount of urea produced. </p><br />
<br />
<br />
<big><b>3.4 Future Work</b></big><br />
<p>As a future work, we will experimentally confirm our results to show that activating the reactions which supply these amino acids is an effective way to increase the production of urea by <i>E. coli</i>.</p><br /><br />
<br />
<big><b>3.5 Reference</big></b><br /><br />
[1] Stefan Schuster, <i>et al.</i> A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic network, Nat Biotechnol(2000) 18:326-32<br /><br />
[2] Mendel Tuchman, <i>et al.</i> Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains, Apple Environ Microbiol(1997) 63: 38-8<br /><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/index.htmTeam:Tokyo Tech/Projects/Urea-cooler/index.htm2011-10-28T10:29:36Z<p>Takuya 1613: </p>
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<ul><br />
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<a href="#abst">1. Abstruct</a><br />
</li><br />
<li><br />
<a href="#2.Gene">2. Genetic Engineering for Urea Production</a><br />
<ul> <br />
<li><a href="#2.1Intro">2.1 Introduction</a></li><br />
<li><a href="#2.2">2.2 Charcterization of rocF and Arg box</a></li><br />
<li><a href="#2.3">2.3 Characterization of Ptrc-RBS-rocF-Arg box</a></li><br />
<li><a href="#2.4">2.4 Use of mutation in argR gene</a></li><br />
</ul><br />
<br />
</li><br />
<li><br />
<a href="#Flux">3. Flux analysis for providing more urea </a><br />
<ul><br />
<li><a href="#abstract">3.1 Abstract</a></li><br />
<li><a href="#intro">3.2 Introduction</a></li><br />
<li><a href="#result">3.3 Results</a></li><br />
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<h1> Urea cooler </h1><br />
<br />
<p><br />
<h2 id="abst">1. Abstract</h2><br />
<p><br />
Coolers can be made by dissolving urea in water because it is an endothermic reaction. So, in order to obtain urea, we made the urea cycle in<i> E.coli</i> by introducing <br />
<i>rocF</i> gene encoding arginase, which is an enzyme that converts L-arginine to L-ornithine and urea.<br />
But just by introducing <i>rocF</i>, <br />
only a little amount of urea can be produced because arginine biosynthesis is repressed by the arginine repressor. Therefore, we tried to derepress arginine biosynthesis in two ways. One is by introducing arginine operator sequences(Arg boxes), which bind the arginine repressor. The other is by using a strain that carries a mutation in a gene which encodes arginine repressor. <br />
Furthermore, we studied elementary flux modes to provide more urea. As a result, <br />
we found out that the artificial urea production system is robust in a stoichiometric point of view. The analysis also <br />
revealed that supplementation of arginine, glutamic acid and aspartic acid would increase urea production rate.<br /><br />
<table style="text-align:center;" align="center"><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/d/de/Characterization_rocF_and_Arg_box.png" alt="Assay data" width="350px" align="center" /></td><br />
<td><img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="350px" /></td><br />
</tr><br />
<tr><br />
<td>Fig.3 Urea concentration in growth media 1 hour after IPTG induction </td><br />
<td> Fig.5 The reactions related with the urea cycle</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<h2 id="2.Gene">2. Genetic Engineering for Urea Production</h2><br />
<h3 id="2.1Intro">2.1 Introduction</h3><br />
<p><br />
Coolers can be made by dissolving urea in water,<br />
since dissolution of urea in water is an endothermic reaction (-57.8 cal/g). So we came up with an idea of creating <i>E. coli</i> that synthesizes urea.<br />
Originally, <i>E. coli</i> has all the enzymes in the urea cycle except for arginase, which converts L-arginine into L-ornithine and urea(Fig.1). so we attempted to complete the urea cycle in <i>E. coli</i> and obtain urea. <br /><br />
<br />
In this work, introduction of the <i>Bacillus subtilis</i> <i>rocF</i> gene which encodes arginase on a <br />
standardized plasmid completed urea cycle and enabled<i> E.coli</i> to produce urea <br />
as reported by TUCHMAN <i>et al</i>.,(1997). <br /><br />
<br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/2/21/TokyoTech_urea-cycle1.png" alt="Urea cycle; Fig1" width="700px" align="center" /><br />
<div class="graph_title"><br />
Fig.1 Addition of a gene which encoding arginase completes urea cycle in <i>E.coli</i>.<br />
</div></center><br /><br />
<br />
<p><br />
However, just by introducing arginase, <i>E. coli</i> produces <br />
only a little amount of urea. TUCHMAN <i>et al</i>. proposed that catabolite <br />
repression in arginine biosynthesis pathway is the main reason for <br />
the low efficiency of production(TUCHMAN <i>et al</i>., 1997). The bacterial <br />
arginine biosynthetic genes are all regulated via a common repressor <br />
protein (arginine repressor) encoded by the <i>argR</i> gene. Arginine represor is activated in the presence of arginine (Fig.2).<br/><br />
TUCHMAN <i>et al</i>. circumvented the repression by introduction of <br />
arginine operator sequences (Arg boxes), which bind the arginine repressor. <br />
With the arginine repressor binding to Arg boxes, the amount of the arginine <br />
repressor which can repress arginine biosynthesis is reduced. <br />
In this work, we tried two ways of derepressing arginine biosynthesis. One way <br />
is introducing the Arg boxes as previous work. The other way is using an <br />
<i>E. coli</i> strain that has an <i>argR</i> deletion genotype so that <br />
the repressor is not synthetized. <br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/3/30/Arginine_repressor.png" alt="Arginine biosynthesis is repressed by arginine repressor in the presence of arginine." align="center" width="600px"/><br />
<div class="graph_title"><br />
Fig.2 Arginine biosynthesis is repressed by arginine repressor in the presence of arginine.<br />
</div><br />
</center><br />
<br />
<h3 id="2.2">2.2 Charcterization of <i>rocF</i> and Arg box</h3><br />
<br />
<p><br />
Introduction of <i>rocF</i> gene on our part <a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a> led to more urea production compared to negative controls. Therefore, we confirmed that insertion of <i>rocF</i> gene resulted in arginase production and completed the urea cycle in <span class="name">E. coli</span> as expected.<br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/d/de/Characterization_rocF_and_Arg_box.png" align="center" width="400px"/></center><br />
<div class="graph_title"><br />
Fig.3 Urea concentration in growth media when <i>rocF</i> or Arg box were introduced strain MG1655. </div></center><br /><br />
<br />
<p><br />
As a way to derepress arginine biosynthesis we introduced Arg box on our part <a href="http://partsregistry.org/Part:BBa_K649401">BBa_K649401</a> in addition to <i>rocF</i> gene (<a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a>) led to production of even more urea. These results show that Arg boxes are effectively derepressing arginine production by deactivating arginine repressor.<br />
</p><br />
<big><b>Materials and Methods</b></big><br /><br />
<p>Materials and methods and other detailed information about this study are shown <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#1.1">here</a>.</p><br />
<h3 id="2.3">2.3 Characterization of Ptrc-RBS-<i>rocF</i>-Arg box</h3><br />
<p><br />
In this study Arg boxes and <i>rocF</i> were introduced separately on high-copy-number plasmids (pSB1C3) and low-copy-number plasmids (pSB3K3). We also tested the effect of Arg boxes when they were introduced downstream of <i>rocF</i> gene on low-copy-number plasmids (pSB3K3 and pSB6A1). Here, Arg boxes didn’t work effectively. This is probably because a low-copy-number plasmid is not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. Detailed information about this study is shown <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#2.1">here</a>.<br />
</p><br />
<br />
<h3 id="2.4">2.4 Use of mutation in <span class="gene">argR</span> gene</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/0/08/RocF_on_pSB3K3.png" align="center" width="400px"/><br />
<div class="graph_title"><br />
Fig.4 Urea concentration in growth media when <i>rocF</i> gene on pSB3K3 was introduced in strain MG1655 (<span class="gene">argR</span> +) or JE6852 (<span class="gene">argR</span> -).<br />
</div><br />
</center><br /><br />
<p><br />
As another way to derepress arginine biosynthesis, we used an <span class="name">E. coli</span> strain that has an <span class="gene">argR</span> deletion genotype (JE6852, This strain was obtained from National Institute of Genetics). In our assay results, urea production in JE6852 is much more than in MG1655 as expected.<br />
</p><br />
<big><b>Methods</b></big><br /><br />
<p><br />
Materials and methods are described <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#2.1">here</a>.<br />
</p><br />
<br />
<h3 id="Flux"> Flux analysis for providing more urea </h3><br />
<h4 id="abstract">3.1 Abstract </h4><br />
<p>This section is about a metabolic engineering study we did about the urea cycle. On the first part we show how we used &ldquo;elementary flux modes&rdquo; (Schuster <i>et al</i>., 2000) to analyze the function of the compounds involved in the urea cycle. Mainly we deduced which compounds act as sources of carbon and sources of nitrogen for the production of urea. On the second part of this study we show how we determined elementary flux modes of the urea cycle to find ways to increase the yield of urea. We focused on a strategy which involves increasing the concentration of four components of the cycle and which we concluded would yield more urea. To confirm our results future experiments will be done.</p><br />
<br />
<div class="graph_title"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="750px" /><br />
<br />Fig.5 The reactions related with the urea cycle<br />
</div><br /><br />
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene"><i>rocF</i></span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.<br /><br />
<br />
<br />
<h5 id="intro">3.2 Introduction</h5><br />
<big><b>3.2.1 What is Elementary Flux Modes?</b></big><br /><br />
<p>In Metabolic Engineering, mathematical modeling is an effective way to increase the products of a reaction. In particular, Flux Analysis, which is based on the hypothesis that the system is in a steady state, is effective to find how to increase these products. The concept of elementary flux mode provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. As a method of metabolic flux analysis, it is based on the hypothesis that the concentration of the reactants and products involved in the cycle does not change. </p><br /><br />
<br />
<big><b>3.2.2 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</b></big><br /><br />
<p>By determining the elementary flux modes of a cycle we can have a more clear view of the function of each of the compounds involved in the cycle being analyzed. Based on the elementary flux modes of the urea cycle, in this study we could deduce that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production and that both L-glutamine and NH<sub>3</sub> act as nitrogen sources for the formation of urea. </p><br /><br />
<cener><br />
<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="200px" /><br />
<span style="font-size:12px;">&rarr;</span><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="200px" /><br />
<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="Fig7" width="400px" /><br />
</center><br /><br />
<br />
<big><b>3.2.3 Finding Modes to Increase the Urea production by <i>E. coli</i></b></big><br />
<p>In this study we determined elementary flux modes to maximize urea production by <i>E. coli</i>. We found that there are two main strategies to increase urea production: one is to increase the amount of carbamoyl phosphate (which formation is known to be the rate-limiting step of the urea cycle). The other one is to increase the concentration of four components of the urea cycle: L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine. We deduced the latter strategy by determining the elementary modes of the urea cycle, and therefore in this study we will focus on the description of this strategy.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="500px" /><br /><br />
Fig.8 Two ways to increase urea production <br /><br /><br />
<br />
<h6 id=results>3.3 Results</h6><br />
<br />
<p>In our study, we considered the enzymatic reactions shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a> to determine the elementary flux modes related to urea production by <i>E. coli</i>. The scheme the overall reaction system is shown in Fig.5 below.</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="600px" /></div><br />
<div class="graph_title"><br />
<br />Fig.5 The reactions related with the urea cycle<br />
</div><br />
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene">rocF</span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.<br /><br />
<br /><br />
<p>By determining the elementary flux modes to produce urea inside <i>E. coli</i>, we found two important results: </p><br />
<p>1. We confirmed both L-glutamine and NH<sub>3</sub> act as nitrogen providers in the urea cycle, as well as deducing that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production. These modes did not make use of organic intermediates. Even though L-glutamine is consumed in order to to transfer the side-chain ammonium group needed for the production of carbamoyl phosphate (which in turn transfers the ammonium group to the urea cycle), free ammonium ion can restore L-glutamine from L-glutamate (which is a byproduct of the reaction that yields carbamoyl phosphate as a product).<br />
</p><br />
<p>2. We concluded that increasing the concentration of L-ornithine will increase the concentration of three related compounds (L-citrulline, N-(L-arginino)succinate, and L-arginine) and this will ultimately lead to an increase in the production of urea. We also noted that since the L-aspartate amino acid, which is needed in the urea cycle we considered(Fig. 5), is normally consumed in protein biosynthesis, so it should be supplied in the culture medium or synthetized by <span class="name">E. coli</span> in order to be able to increase the amount of urea and to maintain the cycles that produce it. </p><br />
<br />
<p>Below is a detailed description of these three results.</p><br />
<big><b>3.3.1 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</b></big><br /><br />
<p>The first step was to determine the flux modes which need of L-glutamine as an input (Mendel <i>et al</i>., 1996). We did this by calculations based on a matrix as the tableau shown below.</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="800px" /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="800px" /><br />
</center><br />
<br /><a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/method">Details about the calculations can be found here</a><br />
<br />
<p>We found eight modes that can produce urea without using organic intermediates. These are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem1">Fig.6</a>. Each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table4">Table 4</a>. In particular, we focused on one the mode displayed in Fig.7.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="fig7" width="750px" /></div><br />
<div class="graph_title"><br />
<div style="font-size:larger"><br />
<b>2NH<sub>3</sub> + HCO<sub>3</sub><sup>-</sup> + 3ATP + H<sub>2</sub>O + NADPH + NAD<sup>+</sup> <br />
→ Urea + 2ADP + AMP + 2Pi + PPi + NADP<sup>+</sup> + NADH</b><br />
</div><br />
Fig.7 One of the urea producing cycles leaded by the concept of elementary flux modes<br />
*The numbers indicate the relative flux carried by the enzymes.<br /><br /><br />
</div><br />
As shown in Fig.7, we deduced that the carbon atom of urea is provided from HCO<sub>3</sub><sup>-</sup> , which is a byproduct of respiration and therefore is already an abundant compound in the bacterial cytoplasm. On the other hand, we also confirmed that carbamoyl phosphate is a nitrogen source for urea production.We also found that the function of L-glutamine in the urea cycle is to provide nitrogen for urea production via carbamoyl phosphate, because ammonium ion can restore L-glutamine from L-glutamate (which is a<br />
byproduct of the reaction that yields carbamoyl phosphate as a product).This conclusion was confirmed experimentally by Mendel <i>et al</i>. (1996). Also, since only providing a nitrogen source is enough to increase urea production by <i>E. coli</i>, we can also conclude that the aritificial urea cycle in <i>E. coli</i> is stoichiometrically well designed. By comparing Fig.5 and Fig.7 we can also observe that, in Fig.7, the reaction which converts L-glutamate to L-ornithine is not needed for urea production. </p><br />
<br />
<big><b>3.3.2. Finding Modes to Increase the Urea production by <i>E. coli</i></b></big><br /><br />
<p>There are two ways to obtain more products from a cycle of reactions: increasing the speed the reactions and increasing the concentration of the reactants. This becomes obvious if we think of the cycle as a track which is travelled by cars (the reactants), and the products as the total sum of the number of laps made by every car. If we double the speed of the cars the number of laps will also double (Fig. 8, lower left). Similarly, if we double the number of cars the number of laps will double as well (Fig. 8, lower right). We applied this analogy to the urea cycle, where the metabolites in the cycle are represented by the cars and the total number of laps represents the total urea yield (as shown in the figure below).<br /><br />
Increasing the velocity of the cars corresponds to increasing the amount of carbamoyl phosphate in the urea cycle, because the reaction which converts L-glutamine to carbamoyl phosphate is the rate-limiting reaction of the cycle. On the other hand, increasing the number of the cars correspond to increasing the concentration of the compounds of the urea cycle. We focused on increasing the concentration the compounds of the urea cycle to find ways to increase the urea yield.<br /><br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="700px" /><br /><br />
Fig.8 Two ways to increase urea production <br /><br /><br />
L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine are four important compounds of the urea cycle. As can be seen I Fig.7, these compounds form a sub-cycle that directly yields urea. Therefore, by increasing the yield of this cycle we can increase the production of urea in <i>E. coli</i>.</p><br />
<br />
<p>We determined the elementary modes which produce these four important compounds. <br />
All elementary flux modes which produce these compounds from L-glutamine or from compounds in TCA cycle produce L-ornithine as intermediate or final product (these modes are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem2">Fig.9</a> and each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table5">Table 5</a>, it can be concluded that increasing the concentration of L-ornithine will increase the production of urea. One of the L-ornithine producing modes is shown in Fig.10.<br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/8/89/Urea-fig11.png" alt="fig11" width="600px" /><br />
</center><br />
<div class="graph_title"><br />
<div style="font-size:larger"><br />
<small><b>2-oxoglutarate + NH<sub>3</sub> + acetyl-CoA + ATP + 3NADPH + 3H<sup>+</sup> <br />
→ L-ornithine + CoASH + acetate + ADP + Pi + H<sub>2</sub>O + 3NADP<sup>+</sup></b></small><br />
</div><br />
Fig.10 One of the L-ornithine producing pathways from intermediates of TCA cycle<br /><br />
*The numbers indicate the relative flux carried by the enzymes.<br /><br /><br />
</div><br />
<br />
<p>The reactions we determined increase the above mentioned four compounds of the urea cycle are shown in Fig. 9. All modes include the reaction that yields L-ornithine by converting L-glutamate to L-ornithine. <br /><br />
We also confirmed that <span class="name">E. coli</span> has no feasible routes for production of these four components other than those indicated in Fig.5. Therefore, we can conclude that the reaction which converts L-glutamate to L-ornithine is a key reaction to increase the reaction rates in the urea cycle and thereby to increase urea production. It should be noted that one of the reactions of the cycle shown in Fig. 5 (the one in the lowest part of the image) requires ATP, NADPH, Acetyl-CoA, and L-glutamate. With the exception of L-glutamate, all of these compounds are already abundant in the cell. Therefore, in future wet experiments, we will focus on studying the effects of supplying L-glutamate to <i>E. coli</i>. We will confirm that by supplying L-glutamate the concentration of intermediates like L-ornithine can be increased and therefore urea production can be increased.<br /><br />
</p><br />
<p>Furthermore, to supply L-glutamine, L-glutamate and L-arginine is effective way to increase the amount of ornithine.(Fig.11)</p><br />
<div arign="center"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a6/Urea_modeling_overview.png" alt="fig.11a" width="600px" /><br /><br />
<span class="graph_title">Fig.11 Ornithine is made from L-glutamine, L-glutamate and L-arginine</span><br />
</div><br />
<p><br />
We also noted that since L-aspartate is consumed in protein biosynthesis, this amino acid should be supplied from in the medium or produced by <i>E. coli</i> itself not only for increasing the amount of urea production, but also for maintaining the cycle.<br /></p><br />
<br />
<p>In conclusion, increasing the concentration of L-glutamine, L-glutamate, L-arginine and L-aspartate is an effective way to increase the amount of urea produced. </p><br />
<br />
<br />
<big><b>3.4 Future Work</b></big><br />
<p>As a future work, we will experimentally confirm our results to show that activating the reactions which supply these amino acids is an effective way to increase the production of urea by <i>E. coli</i>.</p><br /><br />
<br />
<big><b>3.5 Reference</big></b><br /><br />
[1] Stefan Schuster, <i>et al.</i> A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic network, Nat Biotechnol(2000) 18:326-32<br /><br />
[2] Mendel Tuchman, <i>et al.</i> Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains, Apple Environ Microbiol(1997) 63: 38-8<br /><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/index.htmTeam:Tokyo Tech/Projects/Urea-cooler/index.htm2011-10-28T10:29:12Z<p>Takuya 1613: </p>
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<a href="#abst">1. Abstruct</a><br />
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<a href="#2.Gene">2. Genetic Engineering for Urea Production</a><br />
<ul> <br />
<li><a href="#2.1Intro">2.1 Introduction</a></li><br />
<li><a href="#2.2">2.2 Charcterization of rocF and Arg box</a></li><br />
<li><a href="#2.3">2.3 Characterization of Ptrc-RBS-rocF-Arg box</a></li><br />
<li><a href="#2.4">2.4 Use of mutation in argR gene</a></li><br />
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<a href="#Flux">3. Flux analysis for providing more urea </a><br />
<ul><br />
<li><a href="#abstract">3.1 Abstract</a></li><br />
<li><a href="#intro">3.2 Introduction</a></li><br />
<li><a href="#result">3.3 Results</a></li><br />
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<h1> Urea cooler </h1><br />
<br />
<p><br />
<h2 id="abst">1. Abstract</h2><br />
<p><br />
Coolers can be made by dissolving urea in water because it is an endothermic reaction. So, in order to obtain urea, we made the urea cycle in<i> E.coli</i> by introducing <br />
<i>rocF</i> gene encoding arginase, which is an enzyme that converts L-arginine to L-ornithine and urea.<br />
But just by introducing <i>rocF</i>, <br />
only a little amount of urea can be produced because arginine biosynthesis is repressed by the arginine repressor. Therefore, we tried to derepress arginine biosynthesis in two ways. One is by introducing arginine operator sequences(Arg boxes), which bind the arginine repressor. The other is by using a strain that carries a mutation in a gene which encodes arginine repressor. <br />
Furthermore, we studied elementary flux modes to provide more urea. As a result, <br />
we found out that the artificial urea production system is robust in a stoichiometric point of view. The analysis also <br />
revealed that supplementation of arginine, glutamic acid and aspartic acid would increase urea production rate.<br /><br />
<table style="text-align:center;" align="center"><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/d/de/Characterization_rocF_and_Arg_box.png" alt="Assay data" width="350px" align="center" /></td><br />
<td><img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="350px" /></td><br />
</tr><br />
<tr><br />
<td>Fig.3 Urea concentration in growth media 1 hour after IPTG induction </td><br />
<td> Fig.5 The reactions related with the urea cycle</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<h2 id="2.Gene">2. Genetic Engineering for Urea Production</h2><br />
<h3 id="2.1Intro">2.1 Introduction</h3><br />
<p><br />
Coolers can be made by dissolving urea in water,<br />
since dissolution of urea in water is an endothermic reaction (-57.8 cal/g). So we came up with an idea of creating <i>E. coli</i> that synthesizes urea.<br />
Originally, <i>E. coli</i> has all the enzymes in the urea cycle except for arginase, which converts L-arginine into L-ornithine and urea(Fig.1). so we attempted to complete the urea cycle in <i>E. coli</i> and obtain urea. <br /><br />
<br />
In this work, introduction of the <i>Bacillus subtilis</i> <i>rocF</i> gene which encodes arginase on a <br />
standardized plasmid completed urea cycle and enabled<i> E.coli</i> to produce urea <br />
as reported by TUCHMAN <i>et al</i>.,(1997). <br /><br />
<br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/2/21/TokyoTech_urea-cycle1.png" alt="Urea cycle; Fig1" width="700px" align="center" /><br />
<div class="graph_title"><br />
Fig.1 Addition of a gene which encoding arginase completes urea cycle in <i>E.coli</i>.<br />
</div></center><br /><br />
<br />
<p><br />
However, just by introducing arginase, <i>E. coli</i> produces <br />
only a little amount of urea. TUCHMAN <i>et al</i>. proposed that catabolite <br />
repression in arginine biosynthesis pathway is the main reason for <br />
the low efficiency of production(TUCHMAN <i>et al</i>., 1997). The bacterial <br />
arginine biosynthetic genes are all regulated via a common repressor <br />
protein (arginine repressor) encoded by the <i>argR</i> gene. Arginine represor is activated in the presence of arginine (Fig.2).<br/><br />
TUCHMAN <i>et al</i>. circumvented the repression by introduction of <br />
arginine operator sequences (Arg boxes), which bind the arginine repressor. <br />
With the arginine repressor binding to Arg boxes, the amount of the arginine <br />
repressor which can repress arginine biosynthesis is reduced. <br />
In this work, we tried two ways of derepressing arginine biosynthesis. One way <br />
is introducing the Arg boxes as previous work. The other way is using an <br />
<i>E. coli</i> strain that has an <i>argR</i> deletion genotype so that <br />
the repressor is not synthetized. <br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/3/30/Arginine_repressor.png" alt="Arginine biosynthesis is repressed by arginine repressor in the presence of arginine." align="center" width="600px"/><br />
<div class="graph_title"><br />
Fig.2 Arginine biosynthesis is repressed by arginine repressor in the presence of arginine.<br />
</div><br />
</center><br />
<br />
<h3 id="2.2">2.2 Charcterization of <i>rocF</i> and Arg box</h3><br />
<br />
<p><br />
Introduction of <i>rocF</i> gene on our part <a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a> led to more urea production compared to negative controls. Therefore, we confirmed that insertion of <i>rocF</i> gene resulted in arginase production and completed the urea cycle in <span class="name">E. coli</span> as expected.<br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/d/de/Characterization_rocF_and_Arg_box.png" align="center" width="400px"/></center><br />
<div class="graph_title"><br />
Fig.3 Urea concentration in growth media when <i>rocF</i> or Arg box were introduced strain MG1655. </div></center><br /><br />
<br />
<p><br />
As a way to derepress arginine biosynthesis we introduced Arg box on our part <a href="http://partsregistry.org/Part:BBa_K649401">BBa_K649401</a> in addition to <i>rocF</i> gene (<a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a>) led to production of even more urea. These results show that Arg boxes are effectively derepressing arginine production by deactivating arginine repressor.<br />
</p><br />
<big><b>Materials and Methods</b></big><br /><br />
<p>Materials and methods and other detailed information about this study are shown <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#1.1">here</a>.</p><br />
<h3 id="2.3">2.3 Characterization of Ptrc-RBS-<i>rocF</i>-Arg box</h3><br />
<p><br />
In this study Arg boxes and <i>rocF</i> were introduced separately on high-copy-number plasmids (pSB1C3) and low-copy-number plasmids (pSB3K3). We also tested the effect of Arg boxes when they were introduced downstream of <i>rocF</i> gene on low-copy-number plasmids (pSB3K3 and pSB6A1). Here, Arg boxes didn’t work effectively. This is probably because a low-copy-number plasmid is not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. Detailed information about this study is shown <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#2.1">here</a>.<br />
</p><br />
<br />
<h3 id="2.4">2.4 Use of mutation in <span class="gene">argR</span> gene</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/0/08/RocF_on_pSB3K3.png" align="center" width="400px"/><br />
<div class="graph_title"><br />
Fig.4 Urea concentration in growth media when <i>rocF</i> gene on pSB3K3 was introduced in strain MG1655 (<span class="gene">argR</span> +) or JE6852 (<span class="gene">argR</span> -).<br />
</div><br />
</center><br /><br />
<p><br />
As another way to derepress arginine biosynthesis, we used an <span class="name">E. coli</span> strain that has an <span class="gene">argR</span> deletion genotype (JE6852, This strain was obtained from National Institute of Genetics). In our assay results, urea production in JE6852 is much more than in MG1655 as expected.<br />
</p><br />
<big><b>Methods</b></big><br /><br />
<p><br />
Materials and methods are described <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#2.1">here</a>.<br />
</p><br />
<br />
<h3 id="Flux"> Flux analysis for providing more urea </h3><br />
<h4 id="abstract">3.1 Abstract </h4><br />
<p>This section is about a metabolic engineering study we did about the urea cycle. On the first part we show how we used &ldquo;elementary flux modes&rdquo; (Schuster <i>et al</i>., 2000) to analyze the function of the compounds involved in the urea cycle. Mainly we deduced which compounds act as sources of carbon and sources of nitrogen for the production of urea. On the second part of this study we show how we determined elementary flux modes of the urea cycle to find ways to increase the yield of urea. We focused on a strategy which involves increasing the concentration of four components of the cycle and which we concluded would yield more urea. To confirm our results future experiments will be done.</p><br />
<br />
<div class="graph_title"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="750px" /><br />
<br />Fig.5 The reactions related with the urea cycle<br />
</div><br /><br />
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene"><i>rocF</i></span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.<br /><br />
<br />
<br />
<h5 id="intro">3.2 Introduction</h5><br />
<big><b>3.2.1 What is Elementary Flux Modes?</b></big><br /><br />
<p>In Metabolic Engineering, mathematical modeling is an effective way to increase the products of a reaction. In particular, Flux Analysis, which is based on the hypothesis that the system is in a steady state, is effective to find how to increase these products. The concept of elementary flux mode provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. As a method of metabolic flux analysis, it is based on the hypothesis that the concentration of the reactants and products involved in the cycle does not change. </p><br /><br />
<br />
<big><b>3.2.2 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</b></big><br /><br />
<p>By determining the elementary flux modes of a cycle we can have a more clear view of the function of each of the compounds involved in the cycle being analyzed. Based on the elementary flux modes of the urea cycle, in this study we could deduce that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production and that both L-glutamine and NH<sub>3</sub> act as nitrogen sources for the formation of urea. </p><br /><br />
<cener><br />
<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="200px" /><br />
<span style="font-size:12px;">&rarr;</span><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="200px" /><br />
<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="Fig7" width="400px" /><br />
</center><br /><br />
<br />
<big><b>3.2.3 Finding Modes to Increase the Urea production by <i>E. coli</i></b></big><br />
<p>In this study we determined elementary flux modes to maximize urea production by <i>E. coli</i>. We found that there are two main strategies to increase urea production: one is to increase the amount of carbamoyl phosphate (which formation is known to be the rate-limiting step of the urea cycle). The other one is to increase the concentration of four components of the urea cycle: L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine. We deduced the latter strategy by determining the elementary modes of the urea cycle, and therefore in this study we will focus on the description of this strategy.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="500px" /><br /><br />
Fig.8 Two ways to increase urea production <br /><br /><br />
<br />
<h6 id=results>3.3 Results</h6><br />
<br />
<p>In our study, we considered the enzymatic reactions shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a> to determine the elementary flux modes related to urea production by <i>E. coli</i>. The scheme the overall reaction system is shown in Fig.5 below.</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="600px" /></div><br />
<div class="graph_title"><br />
<br />Fig.5 The reactions related with the urea cycle<br />
</div><br />
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene">rocF</span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.<br /><br />
<br /><br />
<p>By determining the elementary flux modes to produce urea inside <i>E. coli</i>, we found two important results: </p><br />
<p>1. We confirmed both L-glutamine and NH<sub>3</sub> act as nitrogen providers in the urea cycle, as well as deducing that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production. These modes did not make use of organic intermediates. Even though L-glutamine is consumed in order to to transfer the side-chain ammonium group needed for the production of carbamoyl phosphate (which in turn transfers the ammonium group to the urea cycle), free ammonium ion can restore L-glutamine from L-glutamate (which is a byproduct of the reaction that yields carbamoyl phosphate as a product).<br />
</p><br />
<p>2. We concluded that increasing the concentration of L-ornithine will increase the concentration of three related compounds (L-citrulline, N-(L-arginino)succinate, and L-arginine) and this will ultimately lead to an increase in the production of urea. We also noted that since the L-aspartate amino acid, which is needed in the urea cycle we considered(Fig. 5), is normally consumed in protein biosynthesis, so it should be supplied in the culture medium or synthetized by <span class="name">E. coli</span> in order to be able to increase the amount of urea and to maintain the cycles that produce it. </p><br />
<br />
<p>Below is a detailed description of these three results.</p><br />
<big><b>3.3.1 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</b></big><br /><br />
<p>The first step was to determine the flux modes which need of L-glutamine as an input (Mendel <i>et al</i>., 1996). We did this by calculations based on a matrix as the tableau shown below.</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="800px" /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="800px" /><br />
</center><br />
<br /><a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/method">Details about the calculations can be found here</a><br />
<br />
<p>We found eight modes that can produce urea without using organic intermediates. These are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem1">Fig.6</a>. Each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table4">Table 4</a>. In particular, we focused on one the mode displayed in Fig.7.<br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="fig7" width="750px" /></div><br />
<div class="graph_title"><br />
<div style="font-size:larger"><br />
<b>2NH<sub>3</sub> + HCO<sub>3</sub><sup>-</sup> + 3ATP + H<sub>2</sub>O + NADPH + NAD<sup>+</sup> <br />
→ Urea + 2ADP + AMP + 2Pi + PPi + NADP<sup>+</sup> + NADH</b><br />
</div><br />
Fig.7 One of the urea producing cycles leaded by the concept of elementary flux modes<br />
*The numbers indicate the relative flux carried by the enzymes.<br /><br /><br />
</div><br />
As shown in Fig.7, we deduced that the carbon atom of urea is provided from HCO<sub>3</sub><sup>-</sup> , which is a byproduct of respiration and therefore is already an abundant compound in the bacterial cytoplasm. On the other hand, we also confirmed that carbamoyl phosphate is a nitrogen source for urea production.We also found that the function of L-glutamine in the urea cycle is to provide nitrogen for urea production via carbamoyl phosphate, because ammonium ion can restore L-glutamine from L-glutamate (which is a<br />
byproduct of the reaction that yields carbamoyl phosphate as a product).This conclusion was confirmed experimentally by Mendel <i>et al</i>. (1996). Also, since only providing a nitrogen source is enough to increase urea production by <i>E. coli</i>, we can also conclude that the aritificial urea cycle in <i>E. coli</i> is stoichiometrically well designed. By comparing Fig.5 and Fig.7 we can also observe that, in Fig.7, the reaction which converts L-glutamate to L-ornithine is not needed for urea production. </p><br />
<br />
<big><b>3.3.2. Finding Modes to Increase the Urea production by <i>E. coli</i></b></big><br /><br />
<p>There are two ways to obtain more products from a cycle of reactions: increasing the speed the reactions and increasing the concentration of the reactants. This becomes obvious if we think of the cycle as a track which is travelled by cars (the reactants), and the products as the total sum of the number of laps made by every car. If we double the speed of the cars the number of laps will also double (Fig. 8, lower left). Similarly, if we double the number of cars the number of laps will double as well (Fig. 8, lower right). We applied this analogy to the urea cycle, where the metabolites in the cycle are represented by the cars and the total number of laps represents the total urea yield (as shown in the figure below).<br /><br />
Increasing the velocity of the cars corresponds to increasing the amount of carbamoyl phosphate in the urea cycle, because the reaction which converts L-glutamine to carbamoyl phosphate is the rate-limiting reaction of the cycle. On the other hand, increasing the number of the cars correspond to increasing the concentration of the compounds of the urea cycle. We focused on increasing the concentration the compounds of the urea cycle to find ways to increase the urea yield.<br /><br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="700px" /><br /><br />
Fig.8 Two ways to increase urea production <br /><br /><br />
L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine are four important compounds of the urea cycle. As can be seen I Fig.7, these compounds form a sub-cycle that directly yields urea. Therefore, by increasing the yield of this cycle we can increase the production of urea in <i>E. coli</i>.</p><br />
<br />
<p>We determined the elementary modes which produce these four important compounds. <br />
All elementary flux modes which produce these compounds from L-glutamine or from compounds in TCA cycle produce L-ornithine as intermediate or final product (these modes are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem2">Fig.9</a> and each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table5">Table 5</a>, it can be concluded that increasing the concentration of L-ornithine will increase the production of urea. One of the L-ornithine producing modes is shown in Fig.10.<br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/8/89/Urea-fig11.png" alt="fig11" width="600px" /><br />
</center><br />
<div class="graph_title"><br />
<div style="font-size:larger"><br />
<small><b>2-oxoglutarate + NH<sub>3</sub> + acetyl-CoA + ATP + 3NADPH + 3H<sup>+</sup> <br />
→ L-ornithine + CoASH + acetate + ADP + Pi + H<sub>2</sub>O + 3NADP<sup>+</sup></b></small><br />
</div><br />
Fig.10 One of the L-ornithine producing pathways from intermediates of TCA cycle<br /><br />
*The numbers indicate the relative flux carried by the enzymes.<br /><br /><br />
</div><br />
<br />
<p>The reactions we determined increase the above mentioned four compounds of the urea cycle are shown in Fig. 9. All modes include the reaction that yields L-ornithine by converting L-glutamate to L-ornithine. <br /><br />
We also confirmed that <span class="name">E. coli</span> has no feasible routes for production of these four components other than those indicated in Fig.5. Therefore, we can conclude that the reaction which converts L-glutamate to L-ornithine is a key reaction to increase the reaction rates in the urea cycle and thereby to increase urea production. It should be noted that one of the reactions of the cycle shown in Fig. 5 (the one in the lowest part of the image) requires ATP, NADPH, Acetyl-CoA, and L-glutamate. With the exception of L-glutamate, all of these compounds are already abundant in the cell. Therefore, in future wet experiments, we will focus on studying the effects of supplying L-glutamate to <i>E. coli</i>. We will confirm that by supplying L-glutamate the concentration of intermediates like L-ornithine can be increased and therefore urea production can be increased.<br /><br />
</p><br />
<p>Furthermore, to supply L-glutamine, L-glutamate and L-arginine is effective way to increase the amount of ornithine.(Fig.11)</p><br />
<div arign="center"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a6/Urea_modeling_overview.png" alt="fig.11a" width="600px" /><br /><br />
<span class="graph_title">Fig.11 Ornithine is made from L-glutamine, L-glutamate and L-arginine</span><br />
</div><br />
<p><br />
We also noted that since L-aspartate is consumed in protein biosynthesis, this amino acid should be supplied from in the medium or produced by <i>E. coli</i> itself not only for increasing the amount of urea production, but also for maintaining the cycle.<br /></p><br />
<br />
<p>In conclusion, increasing the concentration of L-glutamine, L-glutamate, L-arginine and L-aspartate is an effective way to increase the amount of urea produced. </p><br />
<br />
<br />
<big><b>3.4 Future Work</b></big><br />
<p>As a future work, we will experimentally confirm our results to show that activating the reactions which supply these amino acids is an effective way to increase the production of urea by <i>E. coli</i>.</p><br /><br />
<br />
<big><b>3.5 Reference</big></b><br /><br />
[1] Stefan Schuster, <i>et al.</i> A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic network, Nat Biotechnol(2000) 18:326-32<br /><br />
[2] Mendel Tuchman, <i>et al.</i> Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains, Apple Environ Microbiol(1997) 63: 38-8<br /><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htmTeam:Tokyo Tech/Projects/RPS-game/index.htm2011-10-28T10:20:46Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#1">1. The Hands</a></li><br />
<li><br />
<a href="#2">2. The Judges</a><br />
<ul><br />
<li><a href="#2.1">2.1 Using AND-Gate promoters to create Judges</a></li><br />
<li><a href="#2.2">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</a></li><br />
<li><a href="#2.3">2.3 Improving PlsrA</a></li><br />
<li><a href="#2.4">2.4 Improving Plas</a></li><br />
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<a href="#3">3. The Randomizers</a><br />
<ul><br />
<li><a href="#3.1">3.1 Single Colony Isolation</a></li><br />
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<a href="#3.2">3.2 Conditional Knockout by Recombination</a><br />
<ul><br />
<li><a href="#3.2.1">3.2.1 The Requirements</a></li><br />
<li><a href="#3.2.2">3.2.2 The Mechanism</a></li><br />
<li><a href="#3.2.3">3.2.3 Testing the Lox Cassettes</a></li><br />
<li><a href="#3.2.4">3.2.4 Playing Fair: Future Work</a></li><br />
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<a href="#3.3">3.3 Survival of one strain</a><br />
<ul><br />
<li><a href="#3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</a></li><br />
<li><a href="#3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#3.3.4">3.3.4 The Old Model</a></li><br />
<li><a href="#3.3.5">3.3.5 Our New Model</a></li><br />
<li><a href="#3.3.6">3.3.6 The Biological Meaning of our Model</a></li><br />
<li><a href="#3.3.7">3.3.7 Making it Obvious</a></li><br />
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<h1> Rock-Paper-Scissors game </h1><br />
<h2> Introduction </h2><br />
<br />
<br />
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<span class="top"> The Hands </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/1/13/Handzu.png" width="300px" /><br />
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<p><br />
The first step towards making an RPS game that can be played <br />
between humans and bacteria is giving each player a set of <br />
signaling molecules through which they can communicate their <br />
choice of rock, paper or scissors. For that purpose we created <br />
two sets of three signaling molecules corresponding each to rock, paper or scissors. <br />
For humans we used IPTG, aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
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<span class="top"> The Judges </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" width="350px" /><br />
<table><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" width="360px" /></td><br />
<td><img src="http://partsregistry.org/wiki/images/f/f4/LsrA_promoter_activity_introduction.png" width="400px" /></td><br />
</tr><br />
</table><br />
</div><br />
<br />
<p><br />
Although we defined a set of six signaling molecules that can be used to <br />
play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, we designed a set of <br />
<span class="name">E. coli</span> that act as judges. Because there were no working parts related to AI-2 or 3OC12-HSL, we constructed new working lasI promoter, lsrA promoter and LsrR coding gene. <br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Randomizers </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/7/79/Titech-rps-randomizers.png" width="750px" /><br />
</div><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. <br />
To do so, we designed three kinds of randomizers.<br />
</p><br />
<br />
<p><br />
<h2 id="1">1. The Hands</h2><br />
<img src="https://static.igem.org/mediawiki/2011/4/4b/Image001.png" width="600px" style="float:right;"/><br />
<p><br />
The first step towards making an RPS game that can be played between <br />
humans and bacteria is giving each player a set of signaling molecules <br />
through which they can communicate their choice of rock, paper or scissors. <br />
For that purpose we created two sets of three signaling molecules <br />
corresponding each to rock, paper or scissors. For humans we used IPTG, <br />
aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<br />
<h2 id="2" style="clear:both;">2. The Judges</h2><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" alt="the Judge" style="float: left;" /><br />
<p><br />
Although we defined a set of six signaling molecules that can be used <br />
to play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, <br />
we designed a set of <span class="name">E. coli</span> that act as judges. <br />
Each Judge <span class="name">E. coli</span> has an AND-gate promoter and <br />
a fluorescent protein gene that is expressed when the AND-gate promoter <br />
is activated. In this way, the Judge <span class="name">E. coli</span> can <br />
let us know its decision by producing GFP, RFP or CFP to indicate whether <br />
humans win, lose or it is a tie, respectively.<br />
</p><br />
<br />
<h3 id="2.1" style="clear:both;">2.1 Using AND-Gate promoters to create Judges</h3><br />
<p><br />
The first step to make the Judge <span class="name">E. coli</span> was to <br />
find a logic device which could allow the Judge to decide who the winner of the RPS game was. <br />
We found that the AND-gate promoters would fit perfectly for that purpose, <br />
since they can take two signaling molecules as inputs and produce one indicator <br />
as output. Since each of the players has a set of three different signaling <br />
molecules, we need a set of nine Judges, each of which has an AND-gate promoter <br />
that is activated only by one of the nine possible pairs of signaling molecules. <br />
These combinations are shown in the image below.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Tt-image021.png" width="600px" /><br />
</div><br />
<br />
<p><br />
Our next mission was then to check if there were AND-gate promoters BioBricks <br />
that we could use. We searched in the Registry and found a potential AND-gate <br />
promoter designed by iGEM 2007's team Tokyo_Tech. This potential AND-gate <br />
promoter is designed to be activated by the addition of both IPTG and 3OC6-HSL. <br />
However, there was no data showing the IPTG dependency of this promoter, <br />
so we did experiments and confirmed this dependency for the first time in iGEM. <br />
We concluded that the addition of both IPTG and 3OC6-HSL regulates the activity <br />
of this AND-gate promoter. In this way, we completed the construction of one of <br />
the Judges <span class="name">E. coli</span>, which proves in principle that <br />
our game is feasible. To know the detailed method about this assay, <br />
please see <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.">here</a>.<br />
</p><br />
<br />
<div style="float:left"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/BBa_I751101_graph3.png" width="400px" /><br /><br />
<span class="graph_title">Fig 2.1 Tokyo_Tech AND-gate promoter</span><br />
</div><br />
<p><br />
This Plux-lac hybrid promoter contains two LacI operators, a LuxR operator and luxR. <br />
We introduced this part into LacI expressing <span class="name">E. coli</span> strain. <br />
Because IPTG controls the binding of LacI to two LacI-operator parts and 3OC6-HSL <br />
controls the binding of LuxR to a LuxR-operator part, the <span class="gene">gfp</span> <br />
gene activity of the reporter part is dually regulated by IPTG and 3OC6-HSL. <br />
We used promoterless pSB3K3-<span class="gene">gfp</span> (BBa_J54103) as a negative control, <br />
and pAC-P&lambda;-<span class="gene">gfp</span> (chloramphenicol-resistance), which constitutively expressed GFP, <br />
as a positive control. To know about the mechanism of this promoter click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.0.">here</a>.<br />
</p><br /><br />
<br />
<h3 id="2.2" style="clear:both;">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</h3><br />
<br />
<p><br />
In the process of constructing enough AND-gates that could suffice the needs <br />
of our RPS game design, we discovered two faulty BioBricks: lsrA promoter (BBa_K117002) and las promoter (BBa_J64010). <br />
Because of these faulty parts, the Judge <span class="name">E. coli</span> <br />
set we had designed could only sense the Player <span class="name">E. coli</span>'s <br />
signaling molecule 3OC6-HSL (Rock). This ultimately led to an unfair game <br />
because humans could win every time they played with the Paper signaling molecule <br />
(for more on the &ldquo;Sad story of the Rock Player&rdquo; click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/the_sad_stroy_of_the_rock_player">here</a>).<br />
</p><br />
<p><br />
To fix this problem, we improved the old defective las and lsrA promoters parts <br />
by making new parts that work! As can be seen in the experimental information <br />
below (see “Improving lsrA promoter” and “Improving las promoter”), <br />
we confirmed our lasI promoter (<a href="http://partsregistry.org/Part:BBa_K649000">BBa_K649000</a>) <br />
and lsrA promoter (<a href="http://partsregistry.org/Part:BBa_K649100">BBa_K649100</a>) work perfectly!<br />
</p><br />
<p><br />
Since the lsrA promoter plays a key role in the correct functioning of AI-2, <br />
fixing these parts now allows us to use AI-2 as a signaling molecule, which is a <br />
promising advance because of the characteristics of <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.0.">the AI-2 mechanism</a>.<br />
This mechanism prevents AI-2 from cross-talking with other signaling molecules <br />
such as AHL. Hence, this signaling molecule is a very powerful <br />
tool to build complex Synthetic Biology systems.<br />
</p><br />
<p><br />
Finally, one thing we would like outline is that although the promoters we made <br />
are single input promoters, confirmation of their activity is required as a <br />
reference to construct AND gate promoters. Therefore, we solved important issues <br />
and made significant advances towards constructing AND-gate promoters. This allows <br />
<span class="name">E. coli</span> to also choose the signaling molecules <br />
corresponding to Paper and Scissors, so we have again a working RPS game design.<br />
</p><br />
<br />
<h3 id="2.3">2.3 Improving PlsrA</h3><br />
<br />
<div style="float: left;"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4e/PlsrAactivity.png" alt="activity" width="400px" /><br /><br />
<span class="graph_title">Fig 2.2 Not working lsrA promoter(BBa_K117002) <br /> activity and our new lsrA promoter(BBa_K649100)</span> activity<br />
</div><br />
<p><br />
We confirmed that the lsrA promoter (BBa_K117002) does not work properly <br />
(samples used our experiment are listed in Table 2.1 below). The fluorescence intensity of GFP of lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>) was lower <br />
even than those of the negative control (Fig.2.2), which clearly shows that <br />
lsrA promoter(BBa_K117002) does not work as expected. In this experiment, <br />
we measured transcriptional activity of lsrA promoter by introducing a <br />
<span class="gene">gfp</span> gene downstream of this promoter (Fig.2.3).<br />
</p><br /><br /><br /><br /><br />
<table style="clear:both;" border="1" align="center"><br />
<caption>Table 2.1 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="4">JD22597</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB1A2</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 (<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a>)</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 ((<a href="http://partsregistry.org/Part:BBa_K117002">BBa_K117002</a>)-<span class="gene">gfp</span>)</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Lasr3.png" alt="Fig2.3" width="200px" /><br /><br />
<span class="graph_title">Fig2.3 lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>)</span><br />
</div><br />
<br />
<p><br />
To solve this problem, we created the first working iGEM lsrA promoter (BBa_K649100). <br />
Its fluorescence intensity was much higher than that from a promoter-less <br />
<span class="gene">gfp</span> negative control plasmid, showing that our new <br />
lsrA promoter works(Fig2.5). In this experiment, we measured the transcriptional <br />
activity of our lsrA promoter by introducing a <span class="gene">gfp</span> gene downstream of <br />
the promoter(BBa_K649104, Fig2.4). Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.">here</a>.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/ff/Lasr4.png" alt="Fig2.4" width="200px"><br /><br />
<span class="graph_title">Fig 2.4 lsrA promoter-<span class="gene">gfp</span>(BBa_K649104)</span><br />
</div><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/LsrR_repression1.png" alt="Fig4" width="600px"><br /><br />
<span class="graph_title">Fig2.5. LsrR represses lsrA promoter.</span><br />
</div><br />
<br />
<p><br />
Moreover, this promoter can be repressed by our new LsrR part(BBa_K649105). <br />
(samples used our experiments are listed in Table 2.2 below) The fluorescence <br />
intensity of GFP of sample 3 was three times as large as <br />
that of sample 4. This result shows that LsrR successfully repressed <br />
lsrA promoter. In this experiment, we measured LsrR repression activity by <br />
introducing a <span class="gene">gfp</span> gene downstream of lsrA promoter (BBa_K649105, Fig.2.6).<br />
Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#6.">here</a>.<br />
</p><br />
<br />
<table align="center" border="1"><br />
<caption>Table2.2 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="2">JM2.300</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td rowspan="2">MG1655</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span>-PlsrR-<span class="gene">lsrR</span> on pSB3K3</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Lasr7.png" alt="Fig.5" width="200px"><br /><br />
<span class="graph_title">Fig2.6 lsrA promoter-<span class="gene">gfp</span>-lsrR promoter-<span class="gene">lsrR</span>(BBa_K649105)</span><br />
</div><br />
<br />
<h3 id="2.4">2.4 Improving las promoter</h3><br />
<br />
<table align="center"> <br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/8/80/BBa_J64010_graph3.png" align="left" height="330px" /><br />
<span class="graph_title">Fig2.7 (a)</span><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" align="right" height="330px" /><br />
<span class="graph_title">(b)</span><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Fig2.7 (a): Not working lasI promoter (BBa_J64010). <br />
Fig2.7 (b): New working lasI promoter (BBa_K649000) we made. <br />
We confirmed it works as expected. In our assay, we used the same asR <br />
regulator part used in the assay of BBa_ J64010. Clearly, for our <br />
part the fluorescence intensity of 3OC12-HSL+ was higher than that of 3OC12-HSL-.<br /><br />
To know detailed methods about these lasI promoter assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#2.">previous part</a> <br />
and <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3.">new part</a>. <br />
</p><br />
<p><br />
To prove that the LasR regulator used in our PlasI assay works, we did another assay. <br />
Details about this assay can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#1.">here.</a><br />
</p><br />
<br />
<h2 id="3">3. The Randomizers</h2><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. To do so, we designed <br />
three kinds of randomizers: one kind which needs of three types of bacteria <br />
(each of which produces one of the three RPS signaling molecules), <br />
and the other kind that needs of only one type of bacteria which can <br />
synthetize each of the three signaling molecules one at a time and randomly. <br />
Namely, the randomizers are Single Colony Isolation, <br />
Survival of one Strain and Conditional Knockout by Recombination.<br />
</p><br />
<br />
<h3 id="3.1">3.1 Single Colony Isolation</h3><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Image041.png" style="float:right;"/><br />
<p><br />
This is our simplest randomizer design. To make sure <span class="name">E. coli</span> <br />
chooses any of its signaling molecules with equal probability, we put the constructs <br />
for each molecule inside a different bacterium, so we create three types of bacteria: <br />
one synthetizing the corresponding signaling molecule for rock, other synthetizing <br />
the corresponding signaling molecule for paper, and lastly one synthetizing the <br />
corresponding signaling molecule for scissors. By randomly isolating a single colony <br />
out of the many colonies that result from the mixing between the three types of <br />
<span class="name">E. coli</span>, we get a random output as <br />
<span class="name">E. coli</span>'s choice for the RPS game.<br />
</p><br />
<br />
<h3 id="3.2" style="clear:both">3.2 Conditional Knockout by Recombination</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/0c/Cre-lox_mechanism_1.png" width="550px" align="center" /><br />
</div><br />
<p><br />
Our second randomizer differs from our other two randomizers in that all the <br />
three signaling molecules are produced one at a time and randomly by only one type <br />
of bacteria. We were inspired by a paper about &ldquo;brainbow&rdquo; research <br />
on mice to create this randomizer (Livet J <i>et al.</i>, 2007), which is based <br />
on the recombination mechanism of the enzyme Cre and the lox sequences. <br />
We designed a Cre-Lox system which allows <span class="name">E. coli</span> <br />
to express one of its three signaling molecules by means of conditional knockout. <br />
The design is depicted in Fig 1. It should be noted this randomizer is designed <br />
to be used at a single-cell level. When this randomizer is used in groups of cells, <br />
the different signals released by the cells will mix. In this case, by using microfluidic <br />
devices or isolating single colony of bacteria, we can obtain only one of the signaling <br />
molecules produced by the initial group of cells.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/3d/Fig_3.1_-_in_one_cell_1.png" width="700px" align="center" /><br />
</div><br />
<div class="graph_title"><br />
Fig 3.1 - (a) Cre recombinase construction. (b) lox cassettes distribution for the randomizer design<br />
</div><br />
<br />
<h4 id="3.2.1">3.2.1 The Requirements</h4><br />
<br />
<p><br />
Basically, each pair of lox sites (indicated by the same color) mark the points <br />
which the enzyme Cre will excise (they will be cut off the backbone along with the <br />
sequence between them). For a design that allows choosing randomly one of <br />
<span class="name">E. coli</span>’s three signaling molecules, at least two <br />
cassettes of lox sites are needed. When these two cassettes of lox sites and <br />
protein coding sequences are arranged as in Fig3.1(b), <br />
only one signaling molecule is produced.<br />
</p><br />
<p><br />
Our design also required a way to control luxI gene's expression. <br />
To do so, we used an inducible promoter instead of a constitutive promoter. <br />
A constitutive promoter would have caused luxI gene to be expressed beforehand and <br />
could lead to an <span class="name">E. coli</span> producing two signaling molecules <br />
at the same time (the equivalent of showing two hands in the RPS game). In contrast, <br />
the inducible promoter prevents a particular gene from expressing preferentially.<br />
</p> <br />
<p><br />
One last but not less important requirement for our randomizer is that it should <br />
express each of the three signals not only one at a time, but also with the same <br />
probability. This makes the game fair in the sense that <br />
<span class="name">E. coli</span>’s choice in the RPS game is not predictable.<br />
</p><br />
<br />
<h4 id="3.2.2">3.2.2 The Mechanism</h4><br />
<br />
<p><br />
When the blue cassette of Lox sites is excised (Fig 3.1), the signaling molecule <br />
coded by lasI (3OC6-HSL) will be produced. Likewise, when the black Lox cassette <br />
is excised, the signaling molecule coded by luxS (AI-2) will be produced. On the other hand, <br />
a third possible outcome is that recombination does not take place. In this case, <br />
the signaling molecule coded by luxI (3OC6-HSL) will be produced. Also note that excision <br />
of one kind of lox cassette removes the remaining cassette, <br />
thereby preventing further recombination.<br />
</p><br />
<p><br />
As mentioned before, one of the requirements for our randomizer was to have at least <br />
two lox cassettes. This prevents excision of lox sites from different cassettes <br />
(for example one blue lox site and one black lox site). Because the lox71-lox66 cassette and the lox2272-lox2272 cassette are incompatible (Zorana Carter and Daniela Delneri <i>et al.</i>, Yeast 2010), we can use them to build our randomizer.<br />
</p><br />
<br />
<h4 id="3.2.3">3.2.3 Testing the Lox Cassettes</h4><br />
<br />
<p><br />
As stated above, we need two lox cassettes of different recombination frequency <br />
for randomizer. Because there was no working lox parts in registry, we constructed <br />
three original BioBricks for testing whether lox2272 and lox71/66 cassettes work. For <br />
the convenience of testing, fluorescence expressing genes were used in place of signal <br />
molecular expressing genes in construction. After figuring out their working <i>in vitro</i>, we tested them <i>in vivo</i> by detecting red and green fluorescence through fluoro imager and flow cytometer. Furthermore, we compared the relative recombination frequency of two cassettes . Our lox Cassettes constructions <br />
were working properly, and their recombination frequency were different from each other.<br />
</p><br />
<br />
<ul><br />
<li><br />
PlacIQ-lox2272-<span class="gene">gfp</span>-lox2272<a href="http://partsregistry.org/Part:BBa_K649200">(BBa_K649200)</a><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/Lox-gfp-lox_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox2272-<span class="gene">rfp</span>-lox2272-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649201">(BBa_K649201)</a><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Lox2272-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox71-<span class="gene">rfp</span>-lox66-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649202">(BBa_K649202)</a><br />
<img src="https://static.igem.org/mediawiki/2011/b/b4/Lox71-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
</ul><br />
<br />
<p><br />
The <i>in vitro</i> assay with K649200 was made in advance. The preliminary experiment allowed <br />
us to confirm that the Cre-mediated recombination on lox2272 cassette works as designed. <br />
In the assay, Cre recombinase was added to the linear DNA and incubated for 0.5, 2, and 4 hours. <br />
Images of the experiments have been added below.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/6/60/In_vitro_lox_gfp_lox.png" style="float: left;"/><br />
<p><br />
When checking the result by electrophoresis, there were several bands in samples to <br />
which Cre was added(1st, 2nd lane from right which corresponds to 4 hr and 2 hr respectively). <br />
It indicates that excision of the lox sites successfully occurred. <br />
To know detailed about this assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#7."><i>in vitro</i> assay for lox2272</a><br />
</p><br />
<br />
<p><br />
For the <i>in vivo</i> assay, by detecting fluorescenc levels of GFP and mCherry, <br />
we could determine whether recombination occured in K649201 and K649202 and compare <br />
relative recombination frequency between two of them. We prepared a competent cell <br />
JM2.300 into which P<sub>BAD</sub>/araC-Cre(pSB1A2, BBa_I718008) had been constructed. <br />
Subsequently, our BioBrick was constructed into the cell.<br />
</p><br />
<br />
<table border="1" align="center"> <br />
<tr><br />
<th colspan="2">sample</th><br />
<th>arabinose</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">-</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br /> : negative control </td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
</table><br />
<br />
<p><br />
The strain was grown in a 3 mL liquid culture, and 75 &micro;L of 2 M arabinose was added <br />
to induce Cre expression. We used two controls for the experiment. One was the same strain <br />
without arabinose induction, and the other was JM2.300 strain which was induced by arabinose <br />
and had only our BioBrick. All the strains were cultured each for periods of 0.5, 1, 2, and 4 hours, <br />
and in each case the florescence levels were measured by flow cytometer and FLA. <br />
To know the detailed method about this assay, please see here <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8."><i>in vivo</i> assay for lox cassettes</a>.<br />
</p><br />
<br />
<br />
<br />
<p><br />
We confirmed our results optically by taking florescence images. <br />
K649201 transformants with with 0.5 hr-induction of Cre in liquid medium and its two control strains were plated and incubated in 37&deg;C for 12 hours. Images of the three conditions were taken using red <br />
florescence filter, green florescence filter and no filter as shown below, respectively.<br />
</p><br />
<br />
<table align="center"><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/1e/Image146.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(a)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/7/76/Image144.png/800px-Image144.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/2/27/Image142.png/800px-Image142.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(c)</td><br />
</tr><br />
</table><br />
<br />
<p><br />
Fig 3.2 Cre-meditated recombination at lox2272 cassette. Cre-induction period of 0.5 hr<br />
(a)Overlay of Green and Red channel. The leftmost is a negative control which don't have Cre-expressing <br />
plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick K649201. <br />
The rightmost is a uninduced strain which has both plasmid like as the center. <br />
(b)Detection of GFP. The order of samples is same as above. <br />
(c)Detection of mCherry. The order of samples is same as above.<br />
</p><br />
<p><br />
On the sample with the P<sub>BAD</sub>/araC-Cre construction, we found that recombination occurred <br />
when arabinose was added. In contrast to this result, when we measured the levels of the sample <br />
without the P<sub>BAD</sub>/araC-Cre construction, we found that the GFP levels were far lower than <br />
those of the sample with the P<sub>BAD</sub>/araC-Cre construction. This clearly proves that our <br />
lox constructions, both in K649201 and K649202, respond correctly to the effects of Cre recombinase. <br />
A slight detection of green florescence in plate absence of P<sub>BAD</sub>/araC-Cre can be explained<br />
that there happened cross-talk to green channel by FMN(Flavin mononucleotide) or expression of GFP according<br />
to malfunction of terminator before <span class="gene">gfp</span>. We could also observe recombination occurred when arabinose was not <br />
added, which can be explained due to a leaking in the P<sub>BAD</sub>/araC promoter.You can find that K649202 also works well from the images of K649202 on<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8.">here</a>.<br />
</p><br />
<p><br />
Furthermore, we could observe that the arabinose(+) sample of K649202 has higher green/red ratio than that of K649201, which implying the frequency of lox 71/66 casette is higher than that of lox 2272.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/86/111001DK_30_eachpair_111003.png" /><br /><br />
<span class="graph_title">Fig 3.3 Image of six samples of K649201 (up) and K649202 (down) at period of 0.5 hr</span><br />
</div><br />
<br />
<table border="0"> <br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/11/Proportion_2272_0.5hr.png" /></td><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/bb/Proportion_7166_0.5hr.png" /></td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(a)</td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/archive/5/52/20111004160721%21Proportion_area.png" /></td><br />
<td><br />
Fig 3.4 identical plates with Fig 3.3<br /><br />
(a)expression levels of red and green florescence of K649201<br /><br />
(b)expression levels of red and green florescence of K649202<br /><br />
(c)examined area for comparing between red and green florescence at each plate<br /><br />
</td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(c)</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
<p><br />
As we examining green florescence in comparison to red florescence, green expression level was lower than red in <br />
K649201(Fig 3.4(a)), which meaning that considerable plasmids in those cells yet. In contrast, green expression level exceeded red in K649202(Fig 3.4(b)). This result implies that recombination frequency of <br />
lox71/66 cassette is relatively high than that of lox2272 cassette.<br /><br />
<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a2/Flow_cytometer.png"/ width="500px" ></center><br><br />
<p> <span class="graph_title">Fig 3.5 Green fluorescence level of each cell was detected by flow cytometer.<br><br />
(a)arabinose induced strain containing only K649201 and cre-expressing plasmid (b)arabinose supplied strain containing only K649201 (c)arabinose induced starin containing K649202 and cre-expressing plasmid (d)arabinose supplied strain containing only K649202<br><br />
</p><br />
<p><br />
The higher recombination efficiency of lox 71/66 compare to lox2272 was confirmed also by flow cytometer intensity of lox71/66 was higher than that of lox2272, which supports the result detected by FLA. <br />
</span><br />
</p><br />
<br />
<h4 id="3.2.4">3.2.4 Playing Fair: Future Work</h4><br />
<br />
<p><br />
To make each of the outcomes (R, P, and S) equally probable, we are going to quantify the recombination frequency of each lox cassette. This information and adequate Cre induction will be likely to allow us to have an RPS player <br />
<span class="name">E. coli</span> whose choice of either of rock, paper or scissors cannot be predicted.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e6/Regulating_time_1.png" width="700px"/><br />
</div><br />
<br />
<p><br />
In our next experiments, we are going to vary the reaction time and the distance between the lox sites <br />
of each cassette. We believe precise modification of this two parameters must lead to our goal of <br />
making a randomizer in which each of the signaling molecules can be expressed with the same frequency <br />
(which results in each of the outcomes being expressed with the same probability).<br />
</p><br />
<br />
<h3 id="3.3">3.3 Survival of One strain</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" width="500px" /><br />
</div><br />
<br />
<h4 id="3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</h4><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a randomizer <br />
that can be used in our Rock-Paper-Scissors game, due to the fact that only one of the rival strains <br />
will survive. More specifically, we assign to each of the three rival strains either of Rock, Paper or Scissors, <br />
make them compete for survival and take the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h4 id="3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</h4><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in <br />
1996 by Durret and Levin. In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. <br />
However, the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive <br />
(although it can dominate the system, i.e. have the highest population density, for definite periods of time). <br />
We will discuss more on the limitations we found in this model to be adopted as a <br />
randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h4 id="3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</h4><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to outcompete their rivals: <br />
the production of a toxin (a bacteriocin called colicin) that is toxic to other strains, <br />
resistance to the toxin produced by other strains, and a higher birth rate than their rival strains. <br />
Namely, the three types of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) <br />
and colicin-sensitive <span class="name">E. coli</span> (S). <br />
The colicin-producer outcompetes the colicin-sensitive by producing the colicin. The colicin-sensitive <br />
bacteria outcompetes the colicin-resistant because its birth rate is higher than that of the colicin-resistant. <br />
The colicin-resistant outcompetes the colicin producer because its birth rate is higher <br />
than that of the colicin producer. The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" width="500px" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<h4 id="3.3.4">3.3.4 The Old Model</h4><br />
<br />
<p><br />
In the model described by Durret and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). <br />
However, as will be explained afterwards, this results in a loss of <br />
balance that does not allow building a true randomizing system.<br />
</p><br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durret and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
<br />
</div><br />
<br />
<h4 id="3.3.5">3.3.5 Our New Model</h4><br />
<p><br />
As mentioned before, the model proposed by Durret and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it <br />
a biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive <br />
by outcompeting the other two strains, which will die. More specifically, we limited the resistance of the <br />
colicin-resistant bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and <br />
to the sensitive strain, and additionally the resistant strain would also be vulnerable to the colicin produced by <br />
the colicin-producer. Since which strain will be the one that survives is determined by very small differences in <br />
the initial concentrations of the three different populations of bacteria, in practice this systems becomes a <br />
randomizer because of the imprecisions in the measurements that result, for example, when using micropipettes. <br />
This randomizer describes a new competition dynamic that could not be reproduced in the previous model proposed <br />
by Durret and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p><br />
If we set the parameters as follows<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, <br />
we get a graph which clearly shows there are stable points on each of the three axes <br />
(Figure 1, Up).<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
</div><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations <br />
we have set all of the three strains may ultimately survive for infinite peiriods of time. <br />
The differences between our model and the model of Durret and Levin can be seen graphically <br />
in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail conditions <br />
of the model proposed by Durret and Levin (indicated in black font) <br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="3.3.6">3.3.6 The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its <br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in the <br />
production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h4 id="3.3.7">3.3.7 Making it Obvious</h4><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are paths <br />
that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this paths all <br />
have an approximately common origin. In this section we would like to show that the <br />
origin of these paths is practically the same, and that in that sense we have designed <br />
a true randomizer (since, as mentioned before, the imprecisions that result in the <br />
experimental measurements will make it impossible to make the initial <br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three <br />
different strains of E. coli to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<p><br />
We modeled our results using Matlab. <br />
As can be seen in the graphs below, each of the strains can survive if their <br />
initial density in only tree hundredths (a.u.) greater than the other two strains' <br />
initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<h2>References</h2><br />
<p><br />
<ul><br />
<li>Patrick C. Seed et al. "Activation of the Pseudomonas aeruginosa lasI Gene by LasR and the Pseudomonas Autoinducer PAI: an Autoinduction Regulatory Hierarchy" JOURNAL OF BACTERIOLOGY (1995)</li> <br />
<li>Shotaro Ayukawa et al. "Construction of a genetic AND gate under a new standard for assembly of genetic parts" BMC Genomics (2010)</li> <br />
<li>Robert Sidney Cox, III et al. "Programming gene expression with combinatorial promoters" molecular system biology (2007)</li> <br />
<li>Rolf Lutz et al. "Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements" Nucleic Acids Research (1997)</li> <br />
<li>Karina B.Xavier and Bonnie L.Bassler 2004. Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 238-248</li><br />
<li>Ting Xue, Liping Zhao, Haipeng Sun, Xianxuan Zhou, Baolon Sun 2009. LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing. Cell Research (2009), p.1258-1268</li><br />
<li>Liang Wang, Yoshifumi Hashimoto, Chen-Yu Tsao, James J.Valdes, and William E.Bentley 2004. Cyclic AMP(cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 2066-2076</li> <br />
<li>Jean Livet, Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, Robyn A. Bennis, Joshua R. Sanes and Jeff W. Lichtman (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature.</li><br />
<li>Kimi Araki, Masatake Araki1 and Ken-ichi Yamamura (2002). Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites, Nucleic Acids Research</li><br />
<li>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171. </li><br />
<br />
</ul><br />
</p><br />
<br />
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<ul><br />
<li><a href="#1">1. The Hands</a></li><br />
<li><br />
<a href="#2">2. The Judges</a><br />
<ul><br />
<li><a href="#2.1">2.1 Using AND-Gate promoters to create Judges</a></li><br />
<li><a href="#2.2">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</a></li><br />
<li><a href="#2.3">2.3 Improving PlsrA</a></li><br />
<li><a href="#2.4">2.4 Improving Plas</a></li><br />
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<a href="#3">3. The Randomizers</a><br />
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<li><a href="#3.1">3.1 Single Colony Isolation</a></li><br />
<li><br />
<a href="#3.2">3.2 Conditional Knockout by Recombination</a><br />
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<li><a href="#3.2.1">3.2.1 The Requirements</a></li><br />
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<a href="#3.3">3.3 Survival of one strain</a><br />
<ul><br />
<li><a href="#3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</a></li><br />
<li><a href="#3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#3.3.4">3.3.4 The Old Model</a></li><br />
<li><a href="#3.3.5">3.3.5 Our New Model</a></li><br />
<li><a href="#3.3.6">3.3.6 The Biological Meaning of our Model</a></li><br />
<li><a href="#3.3.7">3.3.7 Making it Obvious</a></li><br />
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<h1> Rock-Paper-Scissors game </h1><br />
<h2> Introduction </h2><br />
<br />
<br />
<div align= "center"><br />
<span class="top"> The Hands </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/1/13/Handzu.png" width="300px" /><br />
</div><br />
<p><br />
The first step towards making an RPS game that can be played <br />
between humans and bacteria is giving each player a set of <br />
signaling molecules through which they can communicate their <br />
choice of rock, paper or scissors. For that purpose we created <br />
two sets of three signaling molecules corresponding each to rock, paper or scissors. <br />
For humans we used IPTG, aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Judges </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" width="350px" /><br />
<table><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" width="360px" /></td><br />
<td><img src="http://partsregistry.org/wiki/images/f/f4/LsrA_promoter_activity_introduction.png" width="400px" /></td><br />
</tr><br />
</table><br />
</div><br />
<br />
<p><br />
Although we defined a set of six signaling molecules that can be used to <br />
play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, we designed a set of <br />
<span class="name">E. coli</span> that act as judges. Because there were no working parts related to AI-2 or 3OC12-HSL, we constructed new working lasI promoter, lsrA promoter and LsrR coding gene. <br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Randomizers </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/7/79/Titech-rps-randomizers.png" width="750px" /><br />
</div><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. <br />
To do so, we designed three kinds of randomizers.<br />
</p><br />
<br />
<p><br />
<h2 id="1">1. The Hands</h2><br />
<img src="https://static.igem.org/mediawiki/2011/4/4b/Image001.png" width="600px" style="float:right;"/><br />
<p><br />
The first step towards making an RPS game that can be played between <br />
humans and bacteria is giving each player a set of signaling molecules <br />
through which they can communicate their choice of rock, paper or scissors. <br />
For that purpose we created two sets of three signaling molecules <br />
corresponding each to rock, paper or scissors. For humans we used IPTG, <br />
aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<br />
<h2 id="2" style="clear:both;">2. The Judges</h2><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" alt="the Judge" style="float: left;" /><br />
<p><br />
Although we defined a set of six signaling molecules that can be used <br />
to play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, <br />
we designed a set of <span class="name">E. coli</span> that act as judges. <br />
Each Judge <span class="name">E. coli</span> has an AND-gate promoter and <br />
a fluorescent protein gene that is expressed when the AND-gate promoter <br />
is activated. In this way, the Judge <span class="name">E. coli</span> can <br />
let us know its decision by producing GFP, RFP or CFP to indicate whether <br />
humans win, lose or it is a tie, respectively.<br />
</p><br />
<br />
<h3 id="2.1" style="clear:both;">2.1 Using AND-Gate promoters to create Judges</h3><br />
<p><br />
The first step to make the Judge <span class="name">E. coli</span> was to <br />
find a logic device which could allow the Judge to decide who the winner of the RPS game was. <br />
We found that the AND-gate promoters would fit perfectly for that purpose, <br />
since they can take two signaling molecules as inputs and produce one indicator <br />
as output. Since each of the players has a set of three different signaling <br />
molecules, we need a set of nine Judges, each of which has an AND-gate promoter <br />
that is activated only by one of the nine possible pairs of signaling molecules. <br />
These combinations are shown in the image below.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Tt-image021.png" width="600px" /><br />
</div><br />
<br />
<p><br />
Our next mission was then to check if there were AND-gate promoters BioBricks <br />
that we could use. We searched in the Registry and found a potential AND-gate <br />
promoter designed by iGEM 2007's team Tokyo_Tech. This potential AND-gate <br />
promoter is designed to be activated by the addition of both IPTG and 3OC6-HSL. <br />
However, there was no data showing the IPTG dependency of this promoter, <br />
so we did experiments and confirmed this dependency for the first time in iGEM. <br />
We concluded that the addition of both IPTG and 3OC6-HSL regulates the activity <br />
of this AND-gate promoter. In this way, we completed the construction of one of <br />
the Judges <span class="name">E. coli</span>, which proves in principle that <br />
our game is feasible. To know the detailed method about this assay, <br />
please see <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.">here</a>.<br />
</p><br />
<br />
<div style="float:left"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/BBa_I751101_graph3.png" width="400px" /><br /><br />
<span class="graph_title">Fig 2.1 Tokyo_Tech AND-gate promoter</span><br />
</div><br />
<p><br />
This Plux-lac hybrid promoter contains two LacI operators, a LuxR operator and luxR. <br />
We introduced this part into LacI expressing <span class="name">E. coli</span> strain. <br />
Because IPTG controls the binding of LacI to two LacI-operator parts and 3OC6-HSL <br />
controls the binding of LuxR to a LuxR-operator part, the <span class="gene">gfp</span> <br />
gene activity of the reporter part is dually regulated by IPTG and 3OC6-HSL. <br />
We used promoterless pSB3K3-<span class="gene">gfp</span> (BBa_J54103) as a negative control, <br />
and pAC-P&lambda;-<span class="gene">gfp</span> (chloramphenicol-resistance), which constitutively expressed GFP, <br />
as a positive control. To know about the mechanism of this promoter click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.0.">here</a>.<br />
</p><br /><br />
<br />
<h3 id="2.2" style="clear:both;">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</h3><br />
<br />
<p><br />
In the process of constructing enough AND-gates that could suffice the needs <br />
of our RPS game design, we discovered two faulty BioBricks: lsrA promoter (BBa_K117002) and las promoter (BBa_J64010). <br />
Because of these faulty parts, the Judge <span class="name">E. coli</span> <br />
set we had designed could only sense the Player <span class="name">E. coli</span>'s <br />
signaling molecule 3OC6-HSL (Rock). This ultimately led to an unfair game <br />
because humans could win every time they played with the Paper signaling molecule <br />
(for more on the &ldquo;Sad story of the Rock Player&rdquo; click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/the_sad_stroy_of_the_rock_player">here</a>).<br />
</p><br />
<p><br />
To fix this problem, we improved the old defective las and lsrA promoters parts <br />
by making new parts that work! As can be seen in the experimental information <br />
below (see “Improving lsrA promoter” and “Improving las promoter”), <br />
we confirmed our lasI promoter (<a href="http://partsregistry.org/Part:BBa_K649000">BBa_K649000</a>) <br />
and lsrA promoter (<a href="http://partsregistry.org/Part:BBa_K649100">BBa_K649100</a>) work perfectly!<br />
</p><br />
<p><br />
Since the lsrA promoter plays a key role in the correct functioning of AI-2, <br />
fixing these parts now allows us to use AI-2 as a signaling molecule, which is a <br />
promising advance because of the characteristics of <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.0.">the AI-2 mechanism</a>.<br />
This mechanism prevents AI-2 from cross-talking with other signaling molecules <br />
such as AHL. Hence, this signaling molecule is a very powerful <br />
tool to build complex Synthetic Biology systems.<br />
</p><br />
<p><br />
Finally, one thing we would like outline is that although the promoters we made <br />
are single input promoters, confirmation of their activity is required as a <br />
reference to construct AND gate promoters. Therefore, we solved important issues <br />
and made significant advances towards constructing AND-gate promoters. This allows <br />
<span class="name">E. coli</span> to also choose the signaling molecules <br />
corresponding to Paper and Scissors, so we have again a working RPS game design.<br />
</p><br />
<br />
<h3 id="2.3">2.3 Improving PlsrA</h3><br />
<br />
<div style="float: left;"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4e/PlsrAactivity.png" alt="activity" width="400px" /><br /><br />
<span class="graph_title">Fig 2.2 Not working lsrA promoter(BBa_K117002) <br /> activity and our new lsrA promoter(BBa_K649100)</span> activity<br />
</div><br />
<p><br />
We confirmed that the lsrA promoter (BBa_K117002) does not work properly <br />
(samples used our experiment are listed in Table 2.1 below). The fluorescence intensity of GFP of lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>) was lower <br />
even than those of the negative control (Fig.2.2), which clearly shows that <br />
lsrA promoter(BBa_K117002) does not work as expected. In this experiment, <br />
we measured transcriptional activity of lsrA promoter by introducing a <br />
<span class="gene">gfp</span> gene downstream of this promoter (Fig.2.3).<br />
</p><br /><br /><br /><br /><br />
<table style="clear:both;" border="1" align="center"><br />
<caption>Table 2.1 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="4">JD22597</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB1A2</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 (<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a>)</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 ((<a href="http://partsregistry.org/Part:BBa_K117002">BBa_K117002</a>)-<span class="gene">gfp</span>)</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Lasr3.png" alt="Fig2.3" width="200px" /><br /><br />
<span class="graph_title">Fig2.3 lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>)</span><br />
</div><br />
<br />
<p><br />
To solve this problem, we created the first working iGEM lsrA promoter (BBa_K649100). <br />
Its fluorescence intensity was much higher than that from a promoter-less <br />
<span class="gene">gfp</span> negative control plasmid, showing that our new <br />
lsrA promoter works(Fig2.5). In this experiment, we measured the transcriptional <br />
activity of our lsrA promoter by introducing a <span class="gene">gfp</span> gene downstream of <br />
the promoter(BBa_K649104, Fig2.4). Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.">here</a>.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/ff/Lasr4.png" alt="Fig2.4" width="200px"><br /><br />
<span class="graph_title">Fig 2.4 lsrA promoter-<span class="gene">gfp</span>(BBa_K649104)</span><br />
</div><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/LsrR_repression1.png" alt="Fig4" width="600px"><br /><br />
<span class="graph_title">Fig2.5. LsrR represses lsrA promoter.</span><br />
</div><br />
<br />
<p><br />
Moreover, this promoter can be repressed by our new LsrR part(BBa_K649105). <br />
(samples used our experiments are listed in Table 2.2 below) The fluorescence <br />
intensity of GFP of sample 3 was three times as large as <br />
that of sample 4. This result shows that LsrR successfully repressed <br />
lsrA promoter. In this experiment, we measured LsrR repression activity by <br />
introducing a <span class="gene">gfp</span> gene downstream of lsrA promoter (BBa_K649105, Fig.2.6).<br />
Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#6.">here</a>.<br />
</p><br />
<br />
<table align="center" border="1"><br />
<caption>Table2.2 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="2">JM2.300</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td rowspan="2">MG1655</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span>-PlsrR-<span class="gene">lsrR</span> on pSB3K3</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Lasr7.png" alt="Fig.5" width="200px"><br /><br />
<span class="graph_title">Fig2.6 lsrA promoter-<span class="gene">gfp</span>-lsrR promoter-<span class="gene">lsrR</span>(BBa_K649105)</span><br />
</div><br />
<br />
<h3 id="2.4">2.4 Improving las promoter</h3><br />
<br />
<table align="center"> <br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/8/80/BBa_J64010_graph3.png" align="left" height="330px" /><br />
<span class="graph_title">Fig2.7 (a)</span><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" align="right" height="330px" /><br />
<span class="graph_title">(b)</span><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Fig2.7 (a): Not working lasI promoter (BBa_J64010). <br />
Fig2.7 (b): New working lasI promoter (BBa_K649000) we made. <br />
We confirmed it works as expected. In our assay, we used the same asR <br />
regulator part used in the assay of BBa_ J64010. Clearly, for our <br />
part the fluorescence intensity of 3OC12-HSL+ was higher than that of 3OC12-HSL-.<br /><br />
To know detailed methods about these lasI promoter assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#2.">previous part</a> <br />
and <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3.">new part</a>. <br />
</p><br />
<p><br />
To prove that the LasR regulator used in our PlasI assay works, we did another assay. <br />
Details about this assay can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#1.">here.</a><br />
</p><br />
<br />
<h2 id="3">3. The Randomizers</h2><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. To do so, we designed <br />
three kinds of randomizers: one kind which needs of three types of bacteria <br />
(each of which produces one of the three RPS signaling molecules), <br />
and the other kind that needs of only one type of bacteria which can <br />
synthetize each of the three signaling molecules one at a time and randomly. <br />
Namely, the randomizers are Single Colony Isolation, <br />
Survival of one Strain and Conditional Knockout by Recombination.<br />
</p><br />
<br />
<h3 id="3.1">3.1 Single Colony Isolation</h3><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Image041.png" style="float:right;"/><br />
<p><br />
This is our simplest randomizer design. To make sure <span class="name">E. coli</span> <br />
chooses any of its signaling molecules with equal probability, we put the constructs <br />
for each molecule inside a different bacterium, so we create three types of bacteria: <br />
one synthetizing the corresponding signaling molecule for rock, other synthetizing <br />
the corresponding signaling molecule for paper, and lastly one synthetizing the <br />
corresponding signaling molecule for scissors. By randomly isolating a single colony <br />
out of the many colonies that result from the mixing between the three types of <br />
<span class="name">E. coli</span>, we get a random output as <br />
<span class="name">E. coli</span>'s choice for the RPS game.<br />
</p><br />
<br />
<h3 id="3.2" style="clear:both">3.2 Conditional Knockout by Recombination</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/0c/Cre-lox_mechanism_1.png" width="550px" align="center" /><br />
</div><br />
<p><br />
Our second randomizer differs from our other two randomizers in that all the <br />
three signaling molecules are produced one at a time and randomly by only one type <br />
of bacteria. We were inspired by a paper about &ldquo;brainbow&rdquo; research <br />
on mice to create this randomizer (Livet J <i>et al.</i>, 2007), which is based <br />
on the recombination mechanism of the enzyme Cre and the lox sequences. <br />
We designed a Cre-Lox system which allows <span class="name">E. coli</span> <br />
to express one of its three signaling molecules by means of conditional knockout. <br />
The design is depicted in Fig 1. It should be noted this randomizer is designed <br />
to be used at a single-cell level. When this randomizer is used in groups of cells, <br />
the different signals released by the cells will mix. In this case, by using microfluidic <br />
devices or isolating single colony of bacteria, we can obtain only one of the signaling <br />
molecules produced by the initial group of cells.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/3d/Fig_3.1_-_in_one_cell_1.png" width="700px" align="center" /><br />
</div><br />
<div class="graph_title"><br />
Fig 3.1 - (a) Cre recombinase construction. (b) lox cassettes distribution for the randomizer design<br />
</div><br />
<br />
<h4 id="3.2.1">3.2.1 The Requirements</h4><br />
<br />
<p><br />
Basically, each pair of lox sites (indicated by the same color) mark the points <br />
which the enzyme Cre will excise (they will be cut off the backbone along with the <br />
sequence between them). For a design that allows choosing randomly one of <br />
<span class="name">E. coli</span>’s three signaling molecules, at least two <br />
cassettes of lox sites are needed. When these two cassettes of lox sites and <br />
protein coding sequences are arranged as in Fig3.1(b), <br />
only one signaling molecule is produced.<br />
</p><br />
<p><br />
Our design also required a way to control luxI gene's expression. <br />
To do so, we used an inducible promoter instead of a constitutive promoter. <br />
A constitutive promoter would have caused luxI gene to be expressed beforehand and <br />
could lead to an <span class="name">E. coli</span> producing two signaling molecules <br />
at the same time (the equivalent of showing two hands in the RPS game). In contrast, <br />
the inducible promoter prevents a particular gene from expressing preferentially.<br />
</p> <br />
<p><br />
One last but not less important requirement for our randomizer is that it should <br />
express each of the three signals not only one at a time, but also with the same <br />
probability. This makes the game fair in the sense that <br />
<span class="name">E. coli</span>’s choice in the RPS game is not predictable.<br />
</p><br />
<br />
<h4 id="3.2.2">3.2.2 The Mechanism</h4><br />
<br />
<p><br />
When the blue cassette of Lox sites is excised (Fig 3.1), the signaling molecule <br />
coded by lasI (3OC6-HSL) will be produced. Likewise, when the black Lox cassette <br />
is excised, the signaling molecule coded by luxS (AI-2) will be produced. On the other hand, <br />
a third possible outcome is that recombination does not take place. In this case, <br />
the signaling molecule coded by luxI (3OC6-HSL) will be produced. Also note that excision <br />
of one kind of lox cassette removes the remaining cassette, <br />
thereby preventing further recombination.<br />
</p><br />
<p><br />
As mentioned before, one of the requirements for our randomizer was to have at least <br />
two lox cassettes. This prevents excision of lox sites from different cassettes <br />
(for example one blue lox site and one black lox site). Because the lox71-lox66 cassette and the lox2272-lox2272 cassette are incompatible (Zorana Carter and Daniela Delneri <i>et al.</i>, Yeast 2010), we can use them to build our randomizer.<br />
</p><br />
<br />
<h4 id="3.2.3">3.2.3 Testing the Lox Cassettes</h4><br />
<br />
<p><br />
As stated above, we need two lox cassettes of different recombination frequency <br />
for randomizer. Because there was no working lox parts in registry, we constructed <br />
three original BioBricks for testing whether lox2272 and lox71/66 cassettes work. For <br />
the convenience of testing, fluorescence expressing genes were used in place of signal <br />
molecular expressing genes in construction. After figuring out their working <i>in vitro</i>, we tested them <i>in vivo</i> by detecting red and green fluorescence through fluoro imager and flow cytometer. Furthermore, we compared the relative recombination frequency of two cassettes . Our lox Cassettes constructions <br />
were working properly, and their recombination frequency were different from each other.<br />
</p><br />
<br />
<ul><br />
<li><br />
PlacIQ-lox2272-<span class="gene">gfp</span>-lox2272<a href="http://partsregistry.org/Part:BBa_K649200">(BBa_K649200)</a><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/Lox-gfp-lox_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox2272-<span class="gene">rfp</span>-lox2272-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649201">(BBa_K649201)</a><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Lox2272-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox71-<span class="gene">rfp</span>-lox66-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649202">(BBa_K649202)</a><br />
<img src="https://static.igem.org/mediawiki/2011/b/b4/Lox71-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
</ul><br />
<br />
<p><br />
The <i>in vitro</i> assay with K649200 was made in advance. The preliminary experiment allowed <br />
us to confirm that the Cre-mediated recombination on lox2272 cassette works as designed. <br />
In the assay, Cre recombinase was added to the linear DNA and incubated for 0.5, 2, and 4 hours. <br />
Images of the experiments have been added below.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/6/60/In_vitro_lox_gfp_lox.png" style="float: left;"/><br />
<p><br />
When checking the result by electrophoresis, there were several bands in samples to <br />
which Cre was added(1st, 2nd lane from right which corresponds to 4 hr and 2 hr respectively). <br />
It indicates that excision of the lox sites successfully occurred. <br />
To know detailed about this assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#7."><i>in vitro</i> assay for lox2272</a><br />
</p><br />
<br />
<p><br />
For the <i>in vivo</i> assay, by detecting fluorescenc levels of GFP and mCherry, <br />
we could determine whether recombination occured in K649201 and K649202 and compare <br />
relative recombination frequency between two of them. We prepared a competent cell <br />
JM2.300 into which P<sub>BAD</sub>/araC-Cre(pSB1A2, BBa_I718008) had been constructed. <br />
Subsequently, our BioBrick was constructed into the cell.<br />
</p><br />
<br />
<table border="1" align="center"> <br />
<tr><br />
<th colspan="2">sample</th><br />
<th>arabinose</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">-</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br /> : negative control </td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
</table><br />
<br />
<p><br />
The strain was grown in a 3 mL liquid culture, and 75 &micro;L of 2 M arabinose was added <br />
to induce Cre expression. We used two controls for the experiment. One was the same strain <br />
without arabinose induction, and the other was JM2.300 strain which was induced by arabinose <br />
and had only our BioBrick. All the strains were cultured each for periods of 0.5, 1, 2, and 4 hours, <br />
and in each case the florescence levels were measured by flow cytometer and FLA. <br />
To know the detailed method about this assay, please see here <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8."><i>in vivo</i> assay for lox cassettes</a>.<br />
</p><br />
<br />
<br />
<br />
<p><br />
We confirmed our results optically by taking florescence images. <br />
K649201 transformants with with 0.5 hr-induction of Cre in liquid medium and its two control strains were plated and incubated in 37&deg;C for 12 hours. Images of the three conditions were taken using red <br />
florescence filter, green florescence filter and no filter as shown below, respectively.<br />
</p><br />
<br />
<table align="center"><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/1e/Image146.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(a)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/7/76/Image144.png/800px-Image144.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/2/27/Image142.png/800px-Image142.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(c)</td><br />
</tr><br />
</table><br />
<br />
<p><br />
Fig 3.2 Cre-meditated recombination at lox2272 cassette. Cre-induction period of 0.5 hr<br />
(a)Overlay of Green and Red channel. The leftmost is a negative control which don't have Cre-expressing <br />
plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick K649201. <br />
The rightmost is a uninduced strain which has both plasmid like as the center. <br />
(b)Detection of GFP. The order of samples is same as above. <br />
(c)Detection of mCherry. The order of samples is same as above.<br />
</p><br />
<p><br />
On the sample with the P<sub>BAD</sub>/araC-Cre construction, we found that recombination occurred <br />
when arabinose was added. In contrast to this result, when we measured the levels of the sample <br />
without the P<sub>BAD</sub>/araC-Cre construction, we found that the GFP levels were far lower than <br />
those of the sample with the P<sub>BAD</sub>/araC-Cre construction. This clearly proves that our <br />
lox constructions, both in K649201 and K649202, respond correctly to the effects of Cre recombinase. <br />
A slight detection of green florescence in plate absence of P<sub>BAD</sub>/araC-Cre can be explained<br />
that there happened cross-talk to green channel by FMN(Flavin mononucleotide) or expression of GFP according<br />
to malfunction of terminator before <span class="gene">gfp</span>. We could also observe recombination occurred when arabinose was not <br />
added, which can be explained due to a leaking in the P<sub>BAD</sub>/araC promoter.You can find that K649202 also works well from the images of K649202 on<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8.">here</a>.<br />
</p><br />
<p><br />
Furthermore, we could observe that the arabinose(+) sample of K649202 has higher green/red ratio than that of K649201, which implying the frequency of lox 71/66 casette is higher than that of lox 2272.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/86/111001DK_30_eachpair_111003.png" /><br /><br />
<span class="graph_title">Fig 3.3 Image of six samples of K649201 (up) and K649202 (down) at period of 0.5 hr</span><br />
</div><br />
<br />
<table border="0"> <br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/11/Proportion_2272_0.5hr.png" /></td><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/bb/Proportion_7166_0.5hr.png" /></td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(a)</td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/archive/5/52/20111004160721%21Proportion_area.png" /></td><br />
<td><br />
Fig 3.4 identical plates with Fig 3.3<br /><br />
(a)expression levels of red and green florescence of K649201<br /><br />
(b)expression levels of red and green florescence of K649202<br /><br />
(c)examined area for comparing between red and green florescence at each plate<br /><br />
</td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(c)</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
<p><br />
As we examining green florescence in comparison to red florescence, green expression level was lower than red in <br />
K649201(Fig 3.4(a)), which meaning that considerable plasmids in those cells yet. In contrast, green expression level exceeded red in K649202(Fig 3.4(b)). This result implies that recombination frequency of <br />
lox71/66 cassette is relatively high than that of lox2272 cassette.<br /><br />
<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a2/Flow_cytometer.png"/ width="500px" ></center><br><br />
<p> <span class="graph_title">Fig 3.5 Green fluorescence level of each cell was detected by flow cytometer.<br><br />
(a)arabinose induced strain containing only K649201 and cre-expressing plasmid (b)arabinose supplied strain containing only K649201 (c)arabinose induced starin containing K649202 and cre-expressing plasmid (d)arabinose supplied strain containing only K649202<br><br />
</p><br />
<p><br />
The higher recombination efficiency of lox 71/66 compare to lox2272 was confirmed also by flow cytometer intensity of lox71/66 was higher than that of lox2272, which supports the result detected by FLA. <br />
</span><br />
</p><br />
<br />
<h4 id="3.2.4">3.2.4 Playing Fair: Future Work</h4><br />
<br />
<p><br />
To make each of the outcomes (R, P, and S) equally probable, we are going to quantify the recombination frequency of each lox cassette. This information and adequate Cre induction will be likely to allow us to have an RPS player <br />
<span class="name">E. coli</span> whose choice of either of rock, paper or scissors cannot be predicted.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e6/Regulating_time_1.png" width="700px"/><br />
</div><br />
<br />
<p><br />
In our next experiments, we are going to vary the reaction time and the distance between the lox sites <br />
of each cassette. We believe precise modification of this two parameters must lead to our goal of <br />
making a randomizer in which each of the signaling molecules can be expressed with the same frequency <br />
(which results in each of the outcomes being expressed with the same probability).<br />
</p><br />
<br />
<h3 id="3.3">3.3 Survival of One strain</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" /><br />
</div><br />
<br />
<h4 id="3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</h4><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a randomizer <br />
that can be used in our Rock-Paper-Scissors game, due to the fact that only one of the rival strains <br />
will survive. More specifically, we assign to each of the three rival strains either of Rock, Paper or Scissors, <br />
make them compete for survival and take the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h4 id="3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</h4><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in <br />
1996 by Durret and Levin. In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. <br />
However, the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive <br />
(although it can dominate the system, i.e. have the highest population density, for definite periods of time). <br />
We will discuss more on the limitations we found in this model to be adopted as a <br />
randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h4 id="3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</h4><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to outcompete their rivals: <br />
the production of a toxin (a bacteriocin called colicin) that is toxic to other strains, <br />
resistance to the toxin produced by other strains, and a higher birth rate than their rival strains. <br />
Namely, the three types of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) <br />
and colicin-sensitive <span class="name">E. coli</span> (S). <br />
The colicin-producer outcompetes the colicin-sensitive by producing the colicin. The colicin-sensitive <br />
bacteria outcompetes the colicin-resistant because its birth rate is higher than that of the colicin-resistant. <br />
The colicin-resistant outcompetes the colicin producer because its birth rate is higher <br />
than that of the colicin producer. The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<h4 id="3.3.4">3.3.4 The Old Model</h4><br />
<br />
<p><br />
In the model described by Durret and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). <br />
However, as will be explained afterwards, this results in a loss of <br />
balance that does not allow building a true randomizing system.<br />
</p><br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durret and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
<br />
</div><br />
<br />
<h4 id="3.3.5">3.3.5 Our New Model</h4><br />
<p><br />
As mentioned before, the model proposed by Durret and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it <br />
a biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive <br />
by outcompeting the other two strains, which will die. More specifically, we limited the resistance of the <br />
colicin-resistant bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and <br />
to the sensitive strain, and additionally the resistant strain would also be vulnerable to the colicin produced by <br />
the colicin-producer. Since which strain will be the one that survives is determined by very small differences in <br />
the initial concentrations of the three different populations of bacteria, in practice this systems becomes a <br />
randomizer because of the imprecisions in the measurements that result, for example, when using micropipettes. <br />
This randomizer describes a new competition dynamic that could not be reproduced in the previous model proposed <br />
by Durret and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p><br />
If we set the parameters as follows<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, <br />
we get a graph which clearly shows there are stable points on each of the three axes <br />
(Figure 1, Up).<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
</div><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations <br />
we have set all of the three strains may ultimately survive for infinite peiriods of time. <br />
The differences between our model and the model of Durret and Levin can be seen graphically <br />
in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail conditions <br />
of the model proposed by Durret and Levin (indicated in black font) <br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="3.3.6">3.3.6 The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its <br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in the <br />
production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h4 id="3.3.7">3.3.7 Making it Obvious</h4><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are paths <br />
that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this paths all <br />
have an approximately common origin. In this section we would like to show that the <br />
origin of these paths is practically the same, and that in that sense we have designed <br />
a true randomizer (since, as mentioned before, the imprecisions that result in the <br />
experimental measurements will make it impossible to make the initial <br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three <br />
different strains of E. coli to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<p><br />
We modeled our results using Matlab. <br />
As can be seen in the graphs below, each of the strains can survive if their <br />
initial density in only tree hundredths (a.u.) greater than the other two strains' <br />
initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<h2>References</h2><br />
<p><br />
<ul><br />
<li>Patrick C. Seed et al. "Activation of the Pseudomonas aeruginosa lasI Gene by LasR and the Pseudomonas Autoinducer PAI: an Autoinduction Regulatory Hierarchy" JOURNAL OF BACTERIOLOGY (1995)</li> <br />
<li>Shotaro Ayukawa et al. "Construction of a genetic AND gate under a new standard for assembly of genetic parts" BMC Genomics (2010)</li> <br />
<li>Robert Sidney Cox, III et al. "Programming gene expression with combinatorial promoters" molecular system biology (2007)</li> <br />
<li>Rolf Lutz et al. "Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements" Nucleic Acids Research (1997)</li> <br />
<li>Karina B.Xavier and Bonnie L.Bassler 2004. Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 238-248</li><br />
<li>Ting Xue, Liping Zhao, Haipeng Sun, Xianxuan Zhou, Baolon Sun 2009. LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing. Cell Research (2009), p.1258-1268</li><br />
<li>Liang Wang, Yoshifumi Hashimoto, Chen-Yu Tsao, James J.Valdes, and William E.Bentley 2004. Cyclic AMP(cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 2066-2076</li> <br />
<li>Jean Livet, Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, Robyn A. Bennis, Joshua R. Sanes and Jeff W. Lichtman (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature.</li><br />
<li>Kimi Araki, Masatake Araki1 and Ken-ichi Yamamura (2002). Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites, Nucleic Acids Research</li><br />
<li>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171. </li><br />
<br />
</ul><br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htmTeam:Tokyo Tech/Projects/RPS-game/index.htm2011-10-28T10:15:34Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#1">1. The Hands</a></li><br />
<li><br />
<a href="#2">2. The Judges</a><br />
<ul><br />
<li><a href="#2.1">2.1 Using AND-Gate promoters to create Judges</a></li><br />
<li><a href="#2.2">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</a></li><br />
<li><a href="#2.3">2.3 Improving PlsrA</a></li><br />
<li><a href="#2.4">2.4 Improving Plas</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="#3">3. The Randomizers</a><br />
<ul><br />
<li><a href="#3.1">3.1 Single Colony Isolation</a></li><br />
<li><br />
<a href="#3.2">3.2 Conditional Knockout by Recombination</a><br />
<ul><br />
<li><a href="#3.2.1">3.2.1 The Requirements</a></li><br />
<li><a href="#3.2.2">3.2.2 The Mechanism</a></li><br />
<li><a href="#3.2.3">3.2.3 Testing the Lox Cassettes</a></li><br />
<li><a href="#3.2.4">3.2.4 Playing Fair: Future Work</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="#3.3">3.3 Survival of one strain</a><br />
<ul><br />
<li><a href="#3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</a></li><br />
<li><a href="#3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#3.3.4">3.3.4 The Old Model</a></li><br />
<li><a href="#3.3.5">3.3.5 Our New Model</a></li><br />
<li><a href="#3.3.6">3.3.6 The Biological Meaning of our Model</a></li><br />
<li><a href="#3.3.7">3.3.7 Making it Obvious</a></li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
</div><br />
<br />
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<div class="main"><br />
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<br />
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<h1> Rock-Paper-Scissors game </h1><br />
<h2> Introduction </h2><br />
<br />
<br />
<div align= "center"><br />
<span class="top"> The Hands </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/1/13/Handzu.png" width="300px" /><br />
</div><br />
<p><br />
The first step towards making an RPS game that can be played <br />
between humans and bacteria is giving each player a set of <br />
signaling molecules through which they can communicate their <br />
choice of rock, paper or scissors. For that purpose we created <br />
two sets of three signaling molecules corresponding each to rock, paper or scissors. <br />
For humans we used IPTG, aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Judges </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" width="350px" /><br />
<table><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" width="360px" /></td><br />
<td><img src="http://partsregistry.org/wiki/images/f/f4/LsrA_promoter_activity_introduction.png" width="400px" /></td><br />
</tr><br />
</table><br />
</div><br />
<br />
<p><br />
Although we defined a set of six signaling molecules that can be used to <br />
play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, we designed a set of <br />
<span class="name">E. coli</span> that act as judges. Because there were no working parts related to AI-2 or 3OC12-HSL, we constructed new working lasI promoter, lsrA promoter and LsrR coding gene. <br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Randomizers </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/7/79/Titech-rps-randomizers.png" width="750px" /><br />
</div><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. <br />
To do so, we designed three kinds of randomizers.<br />
</p><br />
<br />
<p><br />
<h2 id="1">1. The Hands</h2><br />
<img src="https://static.igem.org/mediawiki/2011/4/4b/Image001.png" width="600px" style="float:right;"/><br />
<p><br />
The first step towards making an RPS game that can be played between <br />
humans and bacteria is giving each player a set of signaling molecules <br />
through which they can communicate their choice of rock, paper or scissors. <br />
For that purpose we created two sets of three signaling molecules <br />
corresponding each to rock, paper or scissors. For humans we used IPTG, <br />
aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<br />
<h2 id="2" style="clear:both;">2. The Judges</h2><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" alt="the Judge" style="float: left;" /><br />
<p><br />
Although we defined a set of six signaling molecules that can be used <br />
to play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, <br />
we designed a set of <span class="name">E. coli</span> that act as judges. <br />
Each Judge <span class="name">E. coli</span> has an AND-gate promoter and <br />
a fluorescent protein gene that is expressed when the AND-gate promoter <br />
is activated. In this way, the Judge <span class="name">E. coli</span> can <br />
let us know its decision by producing GFP, RFP or CFP to indicate whether <br />
humans win, lose or it is a tie, respectively.<br />
</p><br />
<br />
<h3 id="2.1" style="clear:both;">2.1 Using AND-Gate promoters to create Judges</h3><br />
<p><br />
The first step to make the Judge <span class="name">E. coli</span> was to <br />
find a logic device which could allow the Judge to decide who the winner of the RPS game was. <br />
We found that the AND-gate promoters would fit perfectly for that purpose, <br />
since they can take two signaling molecules as inputs and produce one indicator <br />
as output. Since each of the players has a set of three different signaling <br />
molecules, we need a set of nine Judges, each of which has an AND-gate promoter <br />
that is activated only by one of the nine possible pairs of signaling molecules. <br />
These combinations are shown in the image below.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Tt-image021.png" width="600px" /><br />
</div><br />
<br />
<p><br />
Our next mission was then to check if there were AND-gate promoters BioBricks <br />
that we could use. We searched in the Registry and found a potential AND-gate <br />
promoter designed by iGEM 2007's team Tokyo_Tech. This potential AND-gate <br />
promoter is designed to be activated by the addition of both IPTG and 3OC6-HSL. <br />
However, there was no data showing the IPTG dependency of this promoter, <br />
so we did experiments and confirmed this dependency for the first time in iGEM. <br />
We concluded that the addition of both IPTG and 3OC6-HSL regulates the activity <br />
of this AND-gate promoter. In this way, we completed the construction of one of <br />
the Judges <span class="name">E. coli</span>, which proves in principle that <br />
our game is feasible. To know the detailed method about this assay, <br />
please see <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.">here</a>.<br />
</p><br />
<br />
<div style="float:left"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/BBa_I751101_graph3.png" width="400px" /><br /><br />
<span class="graph_title">Fig 2.1 Tokyo_Tech AND-gate promoter</span><br />
</div><br />
<p><br />
This Plux-lac hybrid promoter contains two LacI operators, a LuxR operator and luxR. <br />
We introduced this part into LacI expressing <span class="name">E. coli</span> strain. <br />
Because IPTG controls the binding of LacI to two LacI-operator parts and 3OC6-HSL <br />
controls the binding of LuxR to a LuxR-operator part, the <span class="gene">gfp</span> <br />
gene activity of the reporter part is dually regulated by IPTG and 3OC6-HSL. <br />
We used promoterless pSB3K3-<span class="gene">gfp</span> (BBa_J54103) as a negative control, <br />
and pAC-P&lambda;-<span class="gene">gfp</span> (chloramphenicol-resistance), which constitutively expressed GFP, <br />
as a positive control. To know about the mechanism of this promoter click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.0.">here</a>.<br />
</p><br /><br />
<br />
<h3 id="2.2" style="clear:both;">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</h3><br />
<br />
<p><br />
In the process of constructing enough AND-gates that could suffice the needs <br />
of our RPS game design, we discovered two faulty BioBricks: lsrA promoter (BBa_K117002) and las promoter (BBa_J64010). <br />
Because of these faulty parts, the Judge <span class="name">E. coli</span> <br />
set we had designed could only sense the Player <span class="name">E. coli</span>'s <br />
signaling molecule 3OC6-HSL (Rock). This ultimately led to an unfair game <br />
because humans could win every time they played with the Paper signaling molecule <br />
(for more on the &ldquo;Sad story of the Rock Player&rdquo; click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/the_sad_stroy_of_the_rock_player">here</a>).<br />
</p><br />
<p><br />
To fix this problem, we improved the old defective las and lsrA promoters parts <br />
by making new parts that work! As can be seen in the experimental information <br />
below (see “Improving lsrA promoter” and “Improving las promoter”), <br />
we confirmed our lasI promoter (<a href="http://partsregistry.org/Part:BBa_K649000">BBa_K649000</a>) <br />
and lsrA promoter (<a href="http://partsregistry.org/Part:BBa_K649100">BBa_K649100</a>) work perfectly!<br />
</p><br />
<p><br />
Since the lsrA promoter plays a key role in the correct functioning of AI-2, <br />
fixing these parts now allows us to use AI-2 as a signaling molecule, which is a <br />
promising advance because of the characteristics of <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.0.">the AI-2 mechanism</a>.<br />
This mechanism prevents AI-2 from cross-talking with other signaling molecules <br />
such as AHL. Hence, this signaling molecule is a very powerful <br />
tool to build complex Synthetic Biology systems.<br />
</p><br />
<p><br />
Finally, one thing we would like outline is that although the promoters we made <br />
are single input promoters, confirmation of their activity is required as a <br />
reference to construct AND gate promoters. Therefore, we solved important issues <br />
and made significant advances towards constructing AND-gate promoters. This allows <br />
<span class="name">E. coli</span> to also choose the signaling molecules <br />
corresponding to Paper and Scissors, so we have again a working RPS game design.<br />
</p><br />
<br />
<h3 id="2.3">2.3 Improving PlsrA</h3><br />
<br />
<div style="float: left;"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4e/PlsrAactivity.png" alt="activity" width="400px" /><br /><br />
<span class="graph_title">Fig 2.2 Not working lsrA promoter(BBa_K117002) <br /> activity and our new lsrA promoter(BBa_K649100)</span> activity<br />
</div><br />
<p><br />
We confirmed that the lsrA promoter (BBa_K117002) does not work properly <br />
(samples used our experiment are listed in Table 2.1 below). The fluorescence intensity of GFP of lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>) was lower <br />
even than those of the negative control (Fig.2.2), which clearly shows that <br />
lsrA promoter(BBa_K117002) does not work as expected. In this experiment, <br />
we measured transcriptional activity of lsrA promoter by introducing a <br />
<span class="gene">gfp</span> gene downstream of this promoter (Fig.2.3).<br />
</p><br /><br /><br />
<table style="clear:both;" border="1" align="center"><br />
<caption>Table 2.1 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="4">JD22597</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB1A2</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 (<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a>)</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 ((<a href="http://partsregistry.org/Part:BBa_K117002">BBa_K117002</a>)-<span class="gene">gfp</span>)</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Lasr3.png" alt="Fig2.3" width="200px" /><br /><br />
<span class="graph_title">Fig2.3 lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>)</span><br />
</div><br />
<br />
<p><br />
To solve this problem, we created the first working iGEM lsrA promoter (BBa_K649100). <br />
Its fluorescence intensity was much higher than that from a promoter-less <br />
<span class="gene">gfp</span> negative control plasmid, showing that our new <br />
lsrA promoter works(Fig2.5). In this experiment, we measured the transcriptional <br />
activity of our lsrA promoter by introducing a <span class="gene">gfp</span> gene downstream of <br />
the promoter(BBa_K649104, Fig2.4). Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.">here</a>.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/ff/Lasr4.png" alt="Fig2.4" width="200px"><br /><br />
<span class="graph_title">Fig 2.4 lsrA promoter-<span class="gene">gfp</span>(BBa_K649104)</span><br />
</div><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/LsrR_repression1.png" alt="Fig4" width="600px"><br /><br />
<span class="graph_title">Fig2.5. LsrR represses lsrA promoter.</span><br />
</div><br />
<br />
<p><br />
Moreover, this promoter can be repressed by our new LsrR part(BBa_K649105). <br />
(samples used our experiments are listed in Table 2.2 below) The fluorescence <br />
intensity of GFP of sample 3 was three times as large as <br />
that of sample 4. This result shows that LsrR successfully repressed <br />
lsrA promoter. In this experiment, we measured LsrR repression activity by <br />
introducing a <span class="gene">gfp</span> gene downstream of lsrA promoter (BBa_K649105, Fig.2.6).<br />
Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#6.">here</a>.<br />
</p><br />
<br />
<table align="center" border="1"><br />
<caption>Table2.2 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="2">JM2.300</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td rowspan="2">MG1655</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span>-PlsrR-<span class="gene">lsrR</span> on pSB3K3</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Lasr7.png" alt="Fig.5" width="200px"><br /><br />
<span class="graph_title">Fig2.6 lsrA promoter-<span class="gene">gfp</span>-lsrR promoter-<span class="gene">lsrR</span>(BBa_K649105)</span><br />
</div><br />
<br />
<h3 id="2.4">2.4 Improving las promoter</h3><br />
<br />
<table align="center"> <br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/8/80/BBa_J64010_graph3.png" align="left" height="330px" /><br />
<span class="graph_title">Fig2.7 (a)</span><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" align="right" height="330px" /><br />
<span class="graph_title">(b)</span><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Fig2.7 (a): Not working lasI promoter (BBa_J64010). <br />
Fig2.7 (b): New working lasI promoter (BBa_K649000) we made. <br />
We confirmed it works as expected. In our assay, we used the same asR <br />
regulator part used in the assay of BBa_ J64010. Clearly, for our <br />
part the fluorescence intensity of 3OC12-HSL+ was higher than that of 3OC12-HSL-.<br /><br />
To know detailed methods about these lasI promoter assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#2.">previous part</a> <br />
and <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3.">new part</a>. <br />
</p><br />
<p><br />
To prove that the LasR regulator used in our PlasI assay works, we did another assay. <br />
Details about this assay can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#1.">here.</a><br />
</p><br />
<br />
<h2 id="3">3. The Randomizers</h2><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. To do so, we designed <br />
three kinds of randomizers: one kind which needs of three types of bacteria <br />
(each of which produces one of the three RPS signaling molecules), <br />
and the other kind that needs of only one type of bacteria which can <br />
synthetize each of the three signaling molecules one at a time and randomly. <br />
Namely, the randomizers are Single Colony Isolation, <br />
Survival of one Strain and Conditional Knockout by Recombination.<br />
</p><br />
<br />
<h3 id="3.1">3.1 Single Colony Isolation</h3><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Image041.png" style="float:right;"/><br />
<p><br />
This is our simplest randomizer design. To make sure <span class="name">E. coli</span> <br />
chooses any of its signaling molecules with equal probability, we put the constructs <br />
for each molecule inside a different bacterium, so we create three types of bacteria: <br />
one synthetizing the corresponding signaling molecule for rock, other synthetizing <br />
the corresponding signaling molecule for paper, and lastly one synthetizing the <br />
corresponding signaling molecule for scissors. By randomly isolating a single colony <br />
out of the many colonies that result from the mixing between the three types of <br />
<span class="name">E. coli</span>, we get a random output as <br />
<span class="name">E. coli</span>'s choice for the RPS game.<br />
</p><br />
<br />
<h3 id="3.2" style="clear:both">3.2 Conditional Knockout by Recombination</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/0c/Cre-lox_mechanism_1.png" width="550px" align="center" /><br />
</div><br />
<p><br />
Our second randomizer differs from our other two randomizers in that all the <br />
three signaling molecules are produced one at a time and randomly by only one type <br />
of bacteria. We were inspired by a paper about &ldquo;brainbow&rdquo; research <br />
on mice to create this randomizer (Livet J <i>et al.</i>, 2007), which is based <br />
on the recombination mechanism of the enzyme Cre and the lox sequences. <br />
We designed a Cre-Lox system which allows <span class="name">E. coli</span> <br />
to express one of its three signaling molecules by means of conditional knockout. <br />
The design is depicted in Fig 1. It should be noted this randomizer is designed <br />
to be used at a single-cell level. When this randomizer is used in groups of cells, <br />
the different signals released by the cells will mix. In this case, by using microfluidic <br />
devices or isolating single colony of bacteria, we can obtain only one of the signaling <br />
molecules produced by the initial group of cells.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/3d/Fig_3.1_-_in_one_cell_1.png" width="700px" align="center" /><br />
</div><br />
<div class="graph_title"><br />
Fig 3.1 - (a) Cre recombinase construction. (b) lox cassettes distribution for the randomizer design<br />
</div><br />
<br />
<h4 id="3.2.1">3.2.1 The Requirements</h4><br />
<br />
<p><br />
Basically, each pair of lox sites (indicated by the same color) mark the points <br />
which the enzyme Cre will excise (they will be cut off the backbone along with the <br />
sequence between them). For a design that allows choosing randomly one of <br />
<span class="name">E. coli</span>’s three signaling molecules, at least two <br />
cassettes of lox sites are needed. When these two cassettes of lox sites and <br />
protein coding sequences are arranged as in Fig3.1(b), <br />
only one signaling molecule is produced.<br />
</p><br />
<p><br />
Our design also required a way to control luxI gene's expression. <br />
To do so, we used an inducible promoter instead of a constitutive promoter. <br />
A constitutive promoter would have caused luxI gene to be expressed beforehand and <br />
could lead to an <span class="name">E. coli</span> producing two signaling molecules <br />
at the same time (the equivalent of showing two hands in the RPS game). In contrast, <br />
the inducible promoter prevents a particular gene from expressing preferentially.<br />
</p> <br />
<p><br />
One last but not less important requirement for our randomizer is that it should <br />
express each of the three signals not only one at a time, but also with the same <br />
probability. This makes the game fair in the sense that <br />
<span class="name">E. coli</span>’s choice in the RPS game is not predictable.<br />
</p><br />
<br />
<h4 id="3.2.2">3.2.2 The Mechanism</h4><br />
<br />
<p><br />
When the blue cassette of Lox sites is excised (Fig 3.1), the signaling molecule <br />
coded by lasI (3OC6-HSL) will be produced. Likewise, when the black Lox cassette <br />
is excised, the signaling molecule coded by luxS (AI-2) will be produced. On the other hand, <br />
a third possible outcome is that recombination does not take place. In this case, <br />
the signaling molecule coded by luxI (3OC6-HSL) will be produced. Also note that excision <br />
of one kind of lox cassette removes the remaining cassette, <br />
thereby preventing further recombination.<br />
</p><br />
<p><br />
As mentioned before, one of the requirements for our randomizer was to have at least <br />
two lox cassettes. This prevents excision of lox sites from different cassettes <br />
(for example one blue lox site and one black lox site). Because the lox71-lox66 cassette and the lox2272-lox2272 cassette are incompatible (Zorana Carter and Daniela Delneri <i>et al.</i>, Yeast 2010), we can use them to build our randomizer.<br />
</p><br />
<br />
<h4 id="3.2.3">3.2.3 Testing the Lox Cassettes</h4><br />
<br />
<p><br />
As stated above, we need two lox cassettes of different recombination frequency <br />
for randomizer. Because there was no working lox parts in registry, we constructed <br />
three original BioBricks for testing whether lox2272 and lox71/66 cassettes work. For <br />
the convenience of testing, fluorescence expressing genes were used in place of signal <br />
molecular expressing genes in construction. After figuring out their working <i>in vitro</i>, we tested them <i>in vivo</i> by detecting red and green fluorescence through fluoro imager and flow cytometer. Furthermore, we compared the relative recombination frequency of two cassettes . Our lox Cassettes constructions <br />
were working properly, and their recombination frequency were different from each other.<br />
</p><br />
<br />
<ul><br />
<li><br />
PlacIQ-lox2272-<span class="gene">gfp</span>-lox2272<a href="http://partsregistry.org/Part:BBa_K649200">(BBa_K649200)</a><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/Lox-gfp-lox_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox2272-<span class="gene">rfp</span>-lox2272-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649201">(BBa_K649201)</a><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Lox2272-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox71-<span class="gene">rfp</span>-lox66-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649202">(BBa_K649202)</a><br />
<img src="https://static.igem.org/mediawiki/2011/b/b4/Lox71-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
</ul><br />
<br />
<p><br />
The <i>in vitro</i> assay with K649200 was made in advance. The preliminary experiment allowed <br />
us to confirm that the Cre-mediated recombination on lox2272 cassette works as designed. <br />
In the assay, Cre recombinase was added to the linear DNA and incubated for 0.5, 2, and 4 hours. <br />
Images of the experiments have been added below.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/6/60/In_vitro_lox_gfp_lox.png" style="float: left;"/><br />
<p><br />
When checking the result by electrophoresis, there were several bands in samples to <br />
which Cre was added(1st, 2nd lane from right which corresponds to 4 hr and 2 hr respectively). <br />
It indicates that excision of the lox sites successfully occurred. <br />
To know detailed about this assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#7."><i>in vitro</i> assay for lox2272</a><br />
</p><br />
<br />
<p><br />
For the <i>in vivo</i> assay, by detecting fluorescenc levels of GFP and mCherry, <br />
we could determine whether recombination occured in K649201 and K649202 and compare <br />
relative recombination frequency between two of them. We prepared a competent cell <br />
JM2.300 into which P<sub>BAD</sub>/araC-Cre(pSB1A2, BBa_I718008) had been constructed. <br />
Subsequently, our BioBrick was constructed into the cell.<br />
</p><br />
<br />
<table border="1" align="center"> <br />
<tr><br />
<th colspan="2">sample</th><br />
<th>arabinose</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">-</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br /> : negative control </td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
</table><br />
<br />
<p><br />
The strain was grown in a 3 mL liquid culture, and 75 &micro;L of 2 M arabinose was added <br />
to induce Cre expression. We used two controls for the experiment. One was the same strain <br />
without arabinose induction, and the other was JM2.300 strain which was induced by arabinose <br />
and had only our BioBrick. All the strains were cultured each for periods of 0.5, 1, 2, and 4 hours, <br />
and in each case the florescence levels were measured by flow cytometer and FLA. <br />
To know the detailed method about this assay, please see here <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8."><i>in vivo</i> assay for lox cassettes</a>.<br />
</p><br />
<br />
<br />
<br />
<p><br />
We confirmed our results optically by taking florescence images. <br />
K649201 transformants with with 0.5 hr-induction of Cre in liquid medium and its two control strains were plated and incubated in 37&deg;C for 12 hours. Images of the three conditions were taken using red <br />
florescence filter, green florescence filter and no filter as shown below, respectively.<br />
</p><br />
<br />
<table align="center"><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/1e/Image146.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(a)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/7/76/Image144.png/800px-Image144.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/2/27/Image142.png/800px-Image142.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(c)</td><br />
</tr><br />
</table><br />
<br />
<p><br />
Fig 3.2 Cre-meditated recombination at lox2272 cassette. Cre-induction period of 0.5 hr<br />
(a)Overlay of Green and Red channel. The leftmost is a negative control which don't have Cre-expressing <br />
plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick K649201. <br />
The rightmost is a uninduced strain which has both plasmid like as the center. <br />
(b)Detection of GFP. The order of samples is same as above. <br />
(c)Detection of mCherry. The order of samples is same as above.<br />
</p><br />
<p><br />
On the sample with the P<sub>BAD</sub>/araC-Cre construction, we found that recombination occurred <br />
when arabinose was added. In contrast to this result, when we measured the levels of the sample <br />
without the P<sub>BAD</sub>/araC-Cre construction, we found that the GFP levels were far lower than <br />
those of the sample with the P<sub>BAD</sub>/araC-Cre construction. This clearly proves that our <br />
lox constructions, both in K649201 and K649202, respond correctly to the effects of Cre recombinase. <br />
A slight detection of green florescence in plate absence of P<sub>BAD</sub>/araC-Cre can be explained<br />
that there happened cross-talk to green channel by FMN(Flavin mononucleotide) or expression of GFP according<br />
to malfunction of terminator before <span class="gene">gfp</span>. We could also observe recombination occurred when arabinose was not <br />
added, which can be explained due to a leaking in the P<sub>BAD</sub>/araC promoter.You can find that K649202 also works well from the images of K649202 on<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8.">here</a>.<br />
</p><br />
<p><br />
Furthermore, we could observe that the arabinose(+) sample of K649202 has higher green/red ratio than that of K649201, which implying the frequency of lox 71/66 casette is higher than that of lox 2272.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/86/111001DK_30_eachpair_111003.png" /><br /><br />
<span class="graph_title">Fig 3.3 Image of six samples of K649201 (up) and K649202 (down) at period of 0.5 hr</span><br />
</div><br />
<br />
<table border="0"> <br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/11/Proportion_2272_0.5hr.png" /></td><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/bb/Proportion_7166_0.5hr.png" /></td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(a)</td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/archive/5/52/20111004160721%21Proportion_area.png" /></td><br />
<td><br />
Fig 3.4 identical plates with Fig 3.3<br /><br />
(a)expression levels of red and green florescence of K649201<br /><br />
(b)expression levels of red and green florescence of K649202<br /><br />
(c)examined area for comparing between red and green florescence at each plate<br /><br />
</td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(c)</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
<p><br />
As we examining green florescence in comparison to red florescence, green expression level was lower than red in <br />
K649201(Fig 3.4(a)), which meaning that considerable plasmids in those cells yet. In contrast, green expression level exceeded red in K649202(Fig 3.4(b)). This result implies that recombination frequency of <br />
lox71/66 cassette is relatively high than that of lox2272 cassette.<br /><br />
<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a2/Flow_cytometer.png"/ width="500px" ></center><br><br />
<p> <span class="graph_title">Fig 3.5 Green fluorescence level of each cell was detected by flow cytometer.<br><br />
(a)arabinose induced strain containing only K649201 and cre-expressing plasmid (b)arabinose supplied strain containing only K649201 (c)arabinose induced starin containing K649202 and cre-expressing plasmid (d)arabinose supplied strain containing only K649202<br><br />
</p><br />
<p><br />
The higher recombination efficiency of lox 71/66 compare to lox2272 was confirmed also by flow cytometer intensity of lox71/66 was higher than that of lox2272, which supports the result detected by FLA. <br />
</span><br />
</p><br />
<br />
<h4 id="3.2.4">3.2.4 Playing Fair: Future Work</h4><br />
<br />
<p><br />
To make each of the outcomes (R, P, and S) equally probable, we are going to quantify the recombination frequency of each lox cassette. This information and adequate Cre induction will be likely to allow us to have an RPS player <br />
<span class="name">E. coli</span> whose choice of either of rock, paper or scissors cannot be predicted.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e6/Regulating_time_1.png" width="700px"/><br />
</div><br />
<br />
<p><br />
In our next experiments, we are going to vary the reaction time and the distance between the lox sites <br />
of each cassette. We believe precise modification of this two parameters must lead to our goal of <br />
making a randomizer in which each of the signaling molecules can be expressed with the same frequency <br />
(which results in each of the outcomes being expressed with the same probability).<br />
</p><br />
<br />
<h3 id="3.3">3.3 Survival of One strain</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" /><br />
</div><br />
<br />
<h4 id="3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</h4><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a randomizer <br />
that can be used in our Rock-Paper-Scissors game, due to the fact that only one of the rival strains <br />
will survive. More specifically, we assign to each of the three rival strains either of Rock, Paper or Scissors, <br />
make them compete for survival and take the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h4 id="3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</h4><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in <br />
1996 by Durret and Levin. In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. <br />
However, the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive <br />
(although it can dominate the system, i.e. have the highest population density, for definite periods of time). <br />
We will discuss more on the limitations we found in this model to be adopted as a <br />
randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h4 id="3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</h4><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to outcompete their rivals: <br />
the production of a toxin (a bacteriocin called colicin) that is toxic to other strains, <br />
resistance to the toxin produced by other strains, and a higher birth rate than their rival strains. <br />
Namely, the three types of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) <br />
and colicin-sensitive <span class="name">E. coli</span> (S). <br />
The colicin-producer outcompetes the colicin-sensitive by producing the colicin. The colicin-sensitive <br />
bacteria outcompetes the colicin-resistant because its birth rate is higher than that of the colicin-resistant. <br />
The colicin-resistant outcompetes the colicin producer because its birth rate is higher <br />
than that of the colicin producer. The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<h4 id="3.3.4">3.3.4 The Old Model</h4><br />
<br />
<p><br />
In the model described by Durret and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). <br />
However, as will be explained afterwards, this results in a loss of <br />
balance that does not allow building a true randomizing system.<br />
</p><br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durret and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
<br />
</div><br />
<br />
<h4 id="3.3.5">3.3.5 Our New Model</h4><br />
<p><br />
As mentioned before, the model proposed by Durret and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it <br />
a biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive <br />
by outcompeting the other two strains, which will die. More specifically, we limited the resistance of the <br />
colicin-resistant bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and <br />
to the sensitive strain, and additionally the resistant strain would also be vulnerable to the colicin produced by <br />
the colicin-producer. Since which strain will be the one that survives is determined by very small differences in <br />
the initial concentrations of the three different populations of bacteria, in practice this systems becomes a <br />
randomizer because of the imprecisions in the measurements that result, for example, when using micropipettes. <br />
This randomizer describes a new competition dynamic that could not be reproduced in the previous model proposed <br />
by Durret and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p><br />
If we set the parameters as follows<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, <br />
we get a graph which clearly shows there are stable points on each of the three axes <br />
(Figure 1, Up).<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
</div><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations <br />
we have set all of the three strains may ultimately survive for infinite peiriods of time. <br />
The differences between our model and the model of Durret and Levin can be seen graphically <br />
in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail conditions <br />
of the model proposed by Durret and Levin (indicated in black font) <br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="3.3.6">3.3.6 The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its <br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in the <br />
production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h4 id="3.3.7">3.3.7 Making it Obvious</h4><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are paths <br />
that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this paths all <br />
have an approximately common origin. In this section we would like to show that the <br />
origin of these paths is practically the same, and that in that sense we have designed <br />
a true randomizer (since, as mentioned before, the imprecisions that result in the <br />
experimental measurements will make it impossible to make the initial <br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three <br />
different strains of E. coli to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<p><br />
We modeled our results using Matlab. <br />
As can be seen in the graphs below, each of the strains can survive if their <br />
initial density in only tree hundredths (a.u.) greater than the other two strains' <br />
initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<h2>References</h2><br />
<p><br />
<ul><br />
<li>Patrick C. Seed et al. "Activation of the Pseudomonas aeruginosa lasI Gene by LasR and the Pseudomonas Autoinducer PAI: an Autoinduction Regulatory Hierarchy" JOURNAL OF BACTERIOLOGY (1995)</li> <br />
<li>Shotaro Ayukawa et al. "Construction of a genetic AND gate under a new standard for assembly of genetic parts" BMC Genomics (2010)</li> <br />
<li>Robert Sidney Cox, III et al. "Programming gene expression with combinatorial promoters" molecular system biology (2007)</li> <br />
<li>Rolf Lutz et al. "Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements" Nucleic Acids Research (1997)</li> <br />
<li>Karina B.Xavier and Bonnie L.Bassler 2004. Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 238-248</li><br />
<li>Ting Xue, Liping Zhao, Haipeng Sun, Xianxuan Zhou, Baolon Sun 2009. LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing. Cell Research (2009), p.1258-1268</li><br />
<li>Liang Wang, Yoshifumi Hashimoto, Chen-Yu Tsao, James J.Valdes, and William E.Bentley 2004. Cyclic AMP(cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 2066-2076</li> <br />
<li>Jean Livet, Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, Robyn A. Bennis, Joshua R. Sanes and Jeff W. Lichtman (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature.</li><br />
<li>Kimi Araki, Masatake Araki1 and Ken-ichi Yamamura (2002). Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites, Nucleic Acids Research</li><br />
<li>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171. </li><br />
<br />
</ul><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htmTeam:Tokyo Tech/Projects/RPS-game/index.htm2011-10-28T10:14:18Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#1">1. The Hands</a></li><br />
<li><br />
<a href="#2">2. The Judges</a><br />
<ul><br />
<li><a href="#2.1">2.1 Using AND-Gate promoters to create Judges</a></li><br />
<li><a href="#2.2">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</a></li><br />
<li><a href="#2.3">2.3 Improving PlsrA</a></li><br />
<li><a href="#2.4">2.4 Improving Plas</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="#3">3. The Randomizers</a><br />
<ul><br />
<li><a href="#3.1">3.1 Single Colony Isolation</a></li><br />
<li><br />
<a href="#3.2">3.2 Conditional Knockout by Recombination</a><br />
<ul><br />
<li><a href="#3.2.1">3.2.1 The Requirements</a></li><br />
<li><a href="#3.2.2">3.2.2 The Mechanism</a></li><br />
<li><a href="#3.2.3">3.2.3 Testing the Lox Cassettes</a></li><br />
<li><a href="#3.2.4">3.2.4 Playing Fair: Future Work</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="#3.3">3.3 Survival of one strain</a><br />
<ul><br />
<li><a href="#3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</a></li><br />
<li><a href="#3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#3.3.4">3.3.4 The Old Model</a></li><br />
<li><a href="#3.3.5">3.3.5 Our New Model</a></li><br />
<li><a href="#3.3.6">3.3.6 The Biological Meaning of our Model</a></li><br />
<li><a href="#3.3.7">3.3.7 Making it Obvious</a></li><br />
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<h1> Rock-Paper-Scissors game </h1><br />
<h2> Introduction </h2><br />
<br />
<br />
<div align= "center"><br />
<span class="top"> The Hands </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/1/13/Handzu.png" width="300px" /><br />
</div><br />
<p><br />
The first step towards making an RPS game that can be played <br />
between humans and bacteria is giving each player a set of <br />
signaling molecules through which they can communicate their <br />
choice of rock, paper or scissors. For that purpose we created <br />
two sets of three signaling molecules corresponding each to rock, paper or scissors. <br />
For humans we used IPTG, aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Judges </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" width="350px" /><br />
<table><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" width="360px" /></td><br />
<td><img src="http://partsregistry.org/wiki/images/f/f4/LsrA_promoter_activity_introduction.png" width="400px" /></td><br />
</tr><br />
</table><br />
</div><br />
<br />
<p><br />
Although we defined a set of six signaling molecules that can be used to <br />
play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, we designed a set of <br />
<span class="name">E. coli</span> that act as judges. Because there were no working parts related to AI-2 or 3OC12-HSL, we constructed new working lasI promoter, lsrA promoter and LsrR coding gene. <br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Randomizers </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/7/79/Titech-rps-randomizers.png" width="750px" /><br />
</div><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. <br />
To do so, we designed three kinds of randomizers.<br />
</p><br />
<br />
<p><br />
<h2 id="1">1. The Hands</h2><br />
<img src="https://static.igem.org/mediawiki/2011/4/4b/Image001.png" width="600px" style="float:right;"/><br />
<p><br />
The first step towards making an RPS game that can be played between <br />
humans and bacteria is giving each player a set of signaling molecules <br />
through which they can communicate their choice of rock, paper or scissors. <br />
For that purpose we created two sets of three signaling molecules <br />
corresponding each to rock, paper or scissors. For humans we used IPTG, <br />
aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<br />
<h2 id="2" style="clear:both;">2. The Judges</h2><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" alt="the Judge" style="float: left;" /><br />
<p><br />
Although we defined a set of six signaling molecules that can be used <br />
to play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, <br />
we designed a set of <span class="name">E. coli</span> that act as judges. <br />
Each Judge <span class="name">E. coli</span> has an AND-gate promoter and <br />
a fluorescent protein gene that is expressed when the AND-gate promoter <br />
is activated. In this way, the Judge <span class="name">E. coli</span> can <br />
let us know its decision by producing GFP, RFP or CFP to indicate whether <br />
humans win, lose or it is a tie, respectively.<br />
</p><br />
<br />
<h3 id="2.1" style="clear:both;">2.1 Using AND-Gate promoters to create Judges</h3><br />
<p><br />
The first step to make the Judge <span class="name">E. coli</span> was to <br />
find a logic device which could allow the Judge to decide who the winner of the RPS game was. <br />
We found that the AND-gate promoters would fit perfectly for that purpose, <br />
since they can take two signaling molecules as inputs and produce one indicator <br />
as output. Since each of the players has a set of three different signaling <br />
molecules, we need a set of nine Judges, each of which has an AND-gate promoter <br />
that is activated only by one of the nine possible pairs of signaling molecules. <br />
These combinations are shown in the image below.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Tt-image021.png" width="600px" /><br />
</div><br />
<br />
<p><br />
Our next mission was then to check if there were AND-gate promoters BioBricks <br />
that we could use. We searched in the Registry and found a potential AND-gate <br />
promoter designed by iGEM 2007's team Tokyo_Tech. This potential AND-gate <br />
promoter is designed to be activated by the addition of both IPTG and 3OC6-HSL. <br />
However, there was no data showing the IPTG dependency of this promoter, <br />
so we did experiments and confirmed this dependency for the first time in iGEM. <br />
We concluded that the addition of both IPTG and 3OC6-HSL regulates the activity <br />
of this AND-gate promoter. In this way, we completed the construction of one of <br />
the Judges <span class="name">E. coli</span>, which proves in principle that <br />
our game is feasible. To know the detailed method about this assay, <br />
please see <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.">here</a>.<br />
</p><br />
<br />
<div style="float:left"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/BBa_I751101_graph3.png" width="400px" /><br /><br />
<span class="graph_title">Fig 2.1 Tokyo_Tech AND-gate promoter</span><br />
</div><br />
<p><br />
This Plux-lac hybrid promoter contains two LacI operators, a LuxR operator and luxR. <br />
We introduced this part into LacI expressing <span class="name">E. coli</span> strain. <br />
Because IPTG controls the binding of LacI to two LacI-operator parts and 3OC6-HSL <br />
controls the binding of LuxR to a LuxR-operator part, the <span class="gene">gfp</span> <br />
gene activity of the reporter part is dually regulated by IPTG and 3OC6-HSL. <br />
We used promoterless pSB3K3-<span class="gene">gfp</span> (BBa_J54103) as a negative control, <br />
and pAC-P&lambda;-<span class="gene">gfp</span> (chloramphenicol-resistance), which constitutively expressed GFP, <br />
as a positive control. To know about the mechanism of this promoter click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.0.">here</a>.<br />
</p><br /><br />
<br />
<h3 id="2.2" style="clear:both;">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</h3><br />
<br />
<p><br />
In the process of constructing enough AND-gates that could suffice the needs <br />
of our RPS game design, we discovered two faulty BioBricks: lsrA promoter (BBa_K117002) and las promoter (BBa_J64010). <br />
Because of these faulty parts, the Judge <span class="name">E. coli</span> <br />
set we had designed could only sense the Player <span class="name">E. coli</span>'s <br />
signaling molecule 3OC6-HSL (Rock). This ultimately led to an unfair game <br />
because humans could win every time they played with the Paper signaling molecule <br />
(for more on the &ldquo;Sad story of the Rock Player&rdquo; click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/the_sad_stroy_of_the_rock_player">here</a>).<br />
</p><br />
<p><br />
To fix this problem, we improved the old defective las and lsrA promoters parts <br />
by making new parts that work! As can be seen in the experimental information <br />
below (see “Improving lsrA promoter” and “Improving las promoter”), <br />
we confirmed our lasI promoter (<a href="http://partsregistry.org/Part:BBa_K649000">BBa_K649000</a>) <br />
and lsrA promoter (<a href="http://partsregistry.org/Part:BBa_K649100">BBa_K649100</a>) work perfectly!<br />
</p><br />
<p><br />
Since the lsrA promoter plays a key role in the correct functioning of AI-2, <br />
fixing these parts now allows us to use AI-2 as a signaling molecule, which is a <br />
promising advance because of the characteristics of <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.0.">the AI-2 mechanism</a>.<br />
This mechanism prevents AI-2 from cross-talking with other signaling molecules <br />
such as AHL. Hence, this signaling molecule is a very powerful <br />
tool to build complex Synthetic Biology systems.<br />
</p><br />
<p><br />
Finally, one thing we would like outline is that although the promoters we made <br />
are single input promoters, confirmation of their activity is required as a <br />
reference to construct AND gate promoters. Therefore, we solved important issues <br />
and made significant advances towards constructing AND-gate promoters. This allows <br />
<span class="name">E. coli</span> to also choose the signaling molecules <br />
corresponding to Paper and Scissors, so we have again a working RPS game design.<br />
</p><br />
<br />
<h3 id="2.3">2.3 Improving PlsrA</h3><br />
<br />
<div style="float: left;"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4e/PlsrAactivity.png" alt="activity" width="400px" /><br /><br />
<span class="graph_title">Fig 2.2 Not working lsrA promoter(BBa_K117002) <br /> activity and our new lsrA promoter(BBa_K649100)</span> activity<br />
</div><br />
<p><br />
We confirmed that the lsrA promoter (BBa_K117002) does not work properly <br />
(samples used our experiment are listed in Table 2.1 below). The fluorescence intensity of GFP of lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>) was lower <br />
even than those of the negative control (Fig.2.2), which clearly shows that <br />
lsrA promoter(BBa_K117002) does not work as expected. In this experiment, <br />
we measured transcriptional activity of lsrA promoter by introducing a <br />
<span class="gene">gfp</span> gene downstream of this promoter (Fig.2.3).<br />
</p><br />
<br /><br />
<table style="clear:both;" border="1" align="center"><br />
<caption>Table 2.1 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="4">JD22597</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB1A2</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 (<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a>)</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 ((<a href="http://partsregistry.org/Part:BBa_K117002">BBa_K117002</a>)-<span class="gene">gfp</span>)</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Lasr3.png" alt="Fig2.3" width="200px" /><br /><br />
<span class="graph_title">Fig2.3 lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>)</span><br />
</div><br />
<br />
<p><br />
To solve this problem, we created the first working iGEM lsrA promoter (BBa_K649100). <br />
Its fluorescence intensity was much higher than that from a promoter-less <br />
<span class="gene">gfp</span> negative control plasmid, showing that our new <br />
lsrA promoter works(Fig2.5). In this experiment, we measured the transcriptional <br />
activity of our lsrA promoter by introducing a <span class="gene">gfp</span> gene downstream of <br />
the promoter(BBa_K649104, Fig2.4). Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.">here</a>.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/ff/Lasr4.png" alt="Fig2.4" width="200px"><br /><br />
<span class="graph_title">Fig 2.4 lsrA promoter-<span class="gene">gfp</span>(BBa_K649104)</span><br />
</div><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/LsrR_repression1.png" alt="Fig4" width="600px"><br /><br />
<span class="graph_title">Fig2.5. LsrR represses lsrA promoter.</span><br />
</div><br />
<br />
<p><br />
Moreover, this promoter can be repressed by our new LsrR part(BBa_K649105). <br />
(samples used our experiments are listed in Table 2.2 below) The fluorescence <br />
intensity of GFP of sample 3 was three times as large as <br />
that of sample 4. This result shows that LsrR successfully repressed <br />
lsrA promoter. In this experiment, we measured LsrR repression activity by <br />
introducing a <span class="gene">gfp</span> gene downstream of lsrA promoter (BBa_K649105, Fig.2.6).<br />
Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#6.">here</a>.<br />
</p><br />
<br />
<table align="center" border="1"><br />
<caption>Table2.2 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="2">JM2.300</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td rowspan="2">MG1655</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span>-PlsrR-<span class="gene">lsrR</span> on pSB3K3</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Lasr7.png" alt="Fig.5" width="200px"><br /><br />
<span class="graph_title">Fig2.6 lsrA promoter-<span class="gene">gfp</span>-lsrR promoter-<span class="gene">lsrR</span>(BBa_K649105)</span><br />
</div><br />
<br />
<h3 id="2.4">2.4 Improving las promoter</h3><br />
<br />
<table align="center"> <br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/8/80/BBa_J64010_graph3.png" align="left" height="330px" /><br />
<span class="graph_title">Fig2.7 (a)</span><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" align="right" height="330px" /><br />
<span class="graph_title">(b)</span><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Fig2.7 (a): Not working lasI promoter (BBa_J64010). <br />
Fig2.7 (b): New working lasI promoter (BBa_K649000) we made. <br />
We confirmed it works as expected. In our assay, we used the same asR <br />
regulator part used in the assay of BBa_ J64010. Clearly, for our <br />
part the fluorescence intensity of 3OC12-HSL+ was higher than that of 3OC12-HSL-.<br /><br />
To know detailed methods about these lasI promoter assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#2.">previous part</a> <br />
and <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3.">new part</a>. <br />
</p><br />
<p><br />
To prove that the LasR regulator used in our PlasI assay works, we did another assay. <br />
Details about this assay can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#1.">here.</a><br />
</p><br />
<br />
<h2 id="3">3. The Randomizers</h2><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. To do so, we designed <br />
three kinds of randomizers: one kind which needs of three types of bacteria <br />
(each of which produces one of the three RPS signaling molecules), <br />
and the other kind that needs of only one type of bacteria which can <br />
synthetize each of the three signaling molecules one at a time and randomly. <br />
Namely, the randomizers are Single Colony Isolation, <br />
Survival of one Strain and Conditional Knockout by Recombination.<br />
</p><br />
<br />
<h3 id="3.1">3.1 Single Colony Isolation</h3><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Image041.png" style="float:right;"/><br />
<p><br />
This is our simplest randomizer design. To make sure <span class="name">E. coli</span> <br />
chooses any of its signaling molecules with equal probability, we put the constructs <br />
for each molecule inside a different bacterium, so we create three types of bacteria: <br />
one synthetizing the corresponding signaling molecule for rock, other synthetizing <br />
the corresponding signaling molecule for paper, and lastly one synthetizing the <br />
corresponding signaling molecule for scissors. By randomly isolating a single colony <br />
out of the many colonies that result from the mixing between the three types of <br />
<span class="name">E. coli</span>, we get a random output as <br />
<span class="name">E. coli</span>'s choice for the RPS game.<br />
</p><br />
<br />
<h3 id="3.2" style="clear:both">3.2 Conditional Knockout by Recombination</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/0c/Cre-lox_mechanism_1.png" width="550px" align="center" /><br />
</div><br />
<p><br />
Our second randomizer differs from our other two randomizers in that all the <br />
three signaling molecules are produced one at a time and randomly by only one type <br />
of bacteria. We were inspired by a paper about &ldquo;brainbow&rdquo; research <br />
on mice to create this randomizer (Livet J <i>et al.</i>, 2007), which is based <br />
on the recombination mechanism of the enzyme Cre and the lox sequences. <br />
We designed a Cre-Lox system which allows <span class="name">E. coli</span> <br />
to express one of its three signaling molecules by means of conditional knockout. <br />
The design is depicted in Fig 1. It should be noted this randomizer is designed <br />
to be used at a single-cell level. When this randomizer is used in groups of cells, <br />
the different signals released by the cells will mix. In this case, by using microfluidic <br />
devices or isolating single colony of bacteria, we can obtain only one of the signaling <br />
molecules produced by the initial group of cells.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/3d/Fig_3.1_-_in_one_cell_1.png" width="700px" align="center" /><br />
</div><br />
<div class="graph_title"><br />
Fig 3.1 - (a) Cre recombinase construction. (b) lox cassettes distribution for the randomizer design<br />
</div><br />
<br />
<h4 id="3.2.1">3.2.1 The Requirements</h4><br />
<br />
<p><br />
Basically, each pair of lox sites (indicated by the same color) mark the points <br />
which the enzyme Cre will excise (they will be cut off the backbone along with the <br />
sequence between them). For a design that allows choosing randomly one of <br />
<span class="name">E. coli</span>’s three signaling molecules, at least two <br />
cassettes of lox sites are needed. When these two cassettes of lox sites and <br />
protein coding sequences are arranged as in Fig3.1(b), <br />
only one signaling molecule is produced.<br />
</p><br />
<p><br />
Our design also required a way to control luxI gene's expression. <br />
To do so, we used an inducible promoter instead of a constitutive promoter. <br />
A constitutive promoter would have caused luxI gene to be expressed beforehand and <br />
could lead to an <span class="name">E. coli</span> producing two signaling molecules <br />
at the same time (the equivalent of showing two hands in the RPS game). In contrast, <br />
the inducible promoter prevents a particular gene from expressing preferentially.<br />
</p> <br />
<p><br />
One last but not less important requirement for our randomizer is that it should <br />
express each of the three signals not only one at a time, but also with the same <br />
probability. This makes the game fair in the sense that <br />
<span class="name">E. coli</span>’s choice in the RPS game is not predictable.<br />
</p><br />
<br />
<h4 id="3.2.2">3.2.2 The Mechanism</h4><br />
<br />
<p><br />
When the blue cassette of Lox sites is excised (Fig 3.1), the signaling molecule <br />
coded by lasI (3OC6-HSL) will be produced. Likewise, when the black Lox cassette <br />
is excised, the signaling molecule coded by luxS (AI-2) will be produced. On the other hand, <br />
a third possible outcome is that recombination does not take place. In this case, <br />
the signaling molecule coded by luxI (3OC6-HSL) will be produced. Also note that excision <br />
of one kind of lox cassette removes the remaining cassette, <br />
thereby preventing further recombination.<br />
</p><br />
<p><br />
As mentioned before, one of the requirements for our randomizer was to have at least <br />
two lox cassettes. This prevents excision of lox sites from different cassettes <br />
(for example one blue lox site and one black lox site). Because the lox71-lox66 cassette and the lox2272-lox2272 cassette are incompatible (Zorana Carter and Daniela Delneri <i>et al.</i>, Yeast 2010), we can use them to build our randomizer.<br />
</p><br />
<br />
<h4 id="3.2.3">3.2.3 Testing the Lox Cassettes</h4><br />
<br />
<p><br />
As stated above, we need two lox cassettes of different recombination frequency <br />
for randomizer. Because there was no working lox parts in registry, we constructed <br />
three original BioBricks for testing whether lox2272 and lox71/66 cassettes work. For <br />
the convenience of testing, fluorescence expressing genes were used in place of signal <br />
molecular expressing genes in construction. After figuring out their working <i>in vitro</i>, we tested them <i>in vivo</i> by detecting red and green fluorescence through fluoro imager and flow cytometer. Furthermore, we compared the relative recombination frequency of two cassettes . Our lox Cassettes constructions <br />
were working properly, and their recombination frequency were different from each other.<br />
</p><br />
<br />
<ul><br />
<li><br />
PlacIQ-lox2272-<span class="gene">gfp</span>-lox2272<a href="http://partsregistry.org/Part:BBa_K649200">(BBa_K649200)</a><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/Lox-gfp-lox_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox2272-<span class="gene">rfp</span>-lox2272-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649201">(BBa_K649201)</a><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Lox2272-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox71-<span class="gene">rfp</span>-lox66-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649202">(BBa_K649202)</a><br />
<img src="https://static.igem.org/mediawiki/2011/b/b4/Lox71-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
</ul><br />
<br />
<p><br />
The <i>in vitro</i> assay with K649200 was made in advance. The preliminary experiment allowed <br />
us to confirm that the Cre-mediated recombination on lox2272 cassette works as designed. <br />
In the assay, Cre recombinase was added to the linear DNA and incubated for 0.5, 2, and 4 hours. <br />
Images of the experiments have been added below.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/6/60/In_vitro_lox_gfp_lox.png" style="float: left;"/><br />
<p><br />
When checking the result by electrophoresis, there were several bands in samples to <br />
which Cre was added(1st, 2nd lane from right which corresponds to 4 hr and 2 hr respectively). <br />
It indicates that excision of the lox sites successfully occurred. <br />
To know detailed about this assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#7."><i>in vitro</i> assay for lox2272</a><br />
</p><br />
<br />
<p><br />
For the <i>in vivo</i> assay, by detecting fluorescenc levels of GFP and mCherry, <br />
we could determine whether recombination occured in K649201 and K649202 and compare <br />
relative recombination frequency between two of them. We prepared a competent cell <br />
JM2.300 into which P<sub>BAD</sub>/araC-Cre(pSB1A2, BBa_I718008) had been constructed. <br />
Subsequently, our BioBrick was constructed into the cell.<br />
</p><br />
<br />
<table border="1" align="center"> <br />
<tr><br />
<th colspan="2">sample</th><br />
<th>arabinose</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">-</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br /> : negative control </td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
</table><br />
<br />
<p><br />
The strain was grown in a 3 mL liquid culture, and 75 &micro;L of 2 M arabinose was added <br />
to induce Cre expression. We used two controls for the experiment. One was the same strain <br />
without arabinose induction, and the other was JM2.300 strain which was induced by arabinose <br />
and had only our BioBrick. All the strains were cultured each for periods of 0.5, 1, 2, and 4 hours, <br />
and in each case the florescence levels were measured by flow cytometer and FLA. <br />
To know the detailed method about this assay, please see here <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8."><i>in vivo</i> assay for lox cassettes</a>.<br />
</p><br />
<br />
<br />
<br />
<p><br />
We confirmed our results optically by taking florescence images. <br />
K649201 transformants with with 0.5 hr-induction of Cre in liquid medium and its two control strains were plated and incubated in 37&deg;C for 12 hours. Images of the three conditions were taken using red <br />
florescence filter, green florescence filter and no filter as shown below, respectively.<br />
</p><br />
<br />
<table align="center"><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/1e/Image146.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(a)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/7/76/Image144.png/800px-Image144.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/2/27/Image142.png/800px-Image142.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(c)</td><br />
</tr><br />
</table><br />
<br />
<p><br />
Fig 3.2 Cre-meditated recombination at lox2272 cassette. Cre-induction period of 0.5 hr<br />
(a)Overlay of Green and Red channel. The leftmost is a negative control which don't have Cre-expressing <br />
plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick K649201. <br />
The rightmost is a uninduced strain which has both plasmid like as the center. <br />
(b)Detection of GFP. The order of samples is same as above. <br />
(c)Detection of mCherry. The order of samples is same as above.<br />
</p><br />
<p><br />
On the sample with the P<sub>BAD</sub>/araC-Cre construction, we found that recombination occurred <br />
when arabinose was added. In contrast to this result, when we measured the levels of the sample <br />
without the P<sub>BAD</sub>/araC-Cre construction, we found that the GFP levels were far lower than <br />
those of the sample with the P<sub>BAD</sub>/araC-Cre construction. This clearly proves that our <br />
lox constructions, both in K649201 and K649202, respond correctly to the effects of Cre recombinase. <br />
A slight detection of green florescence in plate absence of P<sub>BAD</sub>/araC-Cre can be explained<br />
that there happened cross-talk to green channel by FMN(Flavin mononucleotide) or expression of GFP according<br />
to malfunction of terminator before <span class="gene">gfp</span>. We could also observe recombination occurred when arabinose was not <br />
added, which can be explained due to a leaking in the P<sub>BAD</sub>/araC promoter.You can find that K649202 also works well from the images of K649202 on<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8.">here</a>.<br />
</p><br />
<p><br />
Furthermore, we could observe that the arabinose(+) sample of K649202 has higher green/red ratio than that of K649201, which implying the frequency of lox 71/66 casette is higher than that of lox 2272.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/86/111001DK_30_eachpair_111003.png" /><br /><br />
<span class="graph_title">Fig 3.3 Image of six samples of K649201 (up) and K649202 (down) at period of 0.5 hr</span><br />
</div><br />
<br />
<table border="0"> <br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/11/Proportion_2272_0.5hr.png" /></td><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/bb/Proportion_7166_0.5hr.png" /></td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(a)</td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/archive/5/52/20111004160721%21Proportion_area.png" /></td><br />
<td><br />
Fig 3.4 identical plates with Fig 3.3<br /><br />
(a)expression levels of red and green florescence of K649201<br /><br />
(b)expression levels of red and green florescence of K649202<br /><br />
(c)examined area for comparing between red and green florescence at each plate<br /><br />
</td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(c)</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
<p><br />
As we examining green florescence in comparison to red florescence, green expression level was lower than red in <br />
K649201(Fig 3.4(a)), which meaning that considerable plasmids in those cells yet. In contrast, green expression level exceeded red in K649202(Fig 3.4(b)). This result implies that recombination frequency of <br />
lox71/66 cassette is relatively high than that of lox2272 cassette.<br /><br />
<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a2/Flow_cytometer.png"/ width="500px" ></center><br><br />
<p> <span class="graph_title">Fig 3.5 Green fluorescence level of each cell was detected by flow cytometer.<br><br />
(a)arabinose induced strain containing only K649201 and cre-expressing plasmid (b)arabinose supplied strain containing only K649201 (c)arabinose induced starin containing K649202 and cre-expressing plasmid (d)arabinose supplied strain containing only K649202<br><br />
</p><br />
<p><br />
The higher recombination efficiency of lox 71/66 compare to lox2272 was confirmed also by flow cytometer intensity of lox71/66 was higher than that of lox2272, which supports the result detected by FLA. <br />
</span><br />
</p><br />
<br />
<h4 id="3.2.4">3.2.4 Playing Fair: Future Work</h4><br />
<br />
<p><br />
To make each of the outcomes (R, P, and S) equally probable, we are going to quantify the recombination frequency of each lox cassette. This information and adequate Cre induction will be likely to allow us to have an RPS player <br />
<span class="name">E. coli</span> whose choice of either of rock, paper or scissors cannot be predicted.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e6/Regulating_time_1.png" width="700px"/><br />
</div><br />
<br />
<p><br />
In our next experiments, we are going to vary the reaction time and the distance between the lox sites <br />
of each cassette. We believe precise modification of this two parameters must lead to our goal of <br />
making a randomizer in which each of the signaling molecules can be expressed with the same frequency <br />
(which results in each of the outcomes being expressed with the same probability).<br />
</p><br />
<br />
<h3 id="3.3">3.3 Survival of One strain</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" /><br />
</div><br />
<br />
<h4 id="3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</h4><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a randomizer <br />
that can be used in our Rock-Paper-Scissors game, due to the fact that only one of the rival strains <br />
will survive. More specifically, we assign to each of the three rival strains either of Rock, Paper or Scissors, <br />
make them compete for survival and take the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h4 id="3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</h4><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in <br />
1996 by Durret and Levin. In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. <br />
However, the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive <br />
(although it can dominate the system, i.e. have the highest population density, for definite periods of time). <br />
We will discuss more on the limitations we found in this model to be adopted as a <br />
randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h4 id="3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</h4><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to outcompete their rivals: <br />
the production of a toxin (a bacteriocin called colicin) that is toxic to other strains, <br />
resistance to the toxin produced by other strains, and a higher birth rate than their rival strains. <br />
Namely, the three types of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) <br />
and colicin-sensitive <span class="name">E. coli</span> (S). <br />
The colicin-producer outcompetes the colicin-sensitive by producing the colicin. The colicin-sensitive <br />
bacteria outcompetes the colicin-resistant because its birth rate is higher than that of the colicin-resistant. <br />
The colicin-resistant outcompetes the colicin producer because its birth rate is higher <br />
than that of the colicin producer. The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<h4 id="3.3.4">3.3.4 The Old Model</h4><br />
<br />
<p><br />
In the model described by Durret and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). <br />
However, as will be explained afterwards, this results in a loss of <br />
balance that does not allow building a true randomizing system.<br />
</p><br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durret and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
<br />
</div><br />
<br />
<h4 id="3.3.5">3.3.5 Our New Model</h4><br />
<p><br />
As mentioned before, the model proposed by Durret and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it <br />
a biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive <br />
by outcompeting the other two strains, which will die. More specifically, we limited the resistance of the <br />
colicin-resistant bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and <br />
to the sensitive strain, and additionally the resistant strain would also be vulnerable to the colicin produced by <br />
the colicin-producer. Since which strain will be the one that survives is determined by very small differences in <br />
the initial concentrations of the three different populations of bacteria, in practice this systems becomes a <br />
randomizer because of the imprecisions in the measurements that result, for example, when using micropipettes. <br />
This randomizer describes a new competition dynamic that could not be reproduced in the previous model proposed <br />
by Durret and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p><br />
If we set the parameters as follows<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, <br />
we get a graph which clearly shows there are stable points on each of the three axes <br />
(Figure 1, Up).<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
</div><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations <br />
we have set all of the three strains may ultimately survive for infinite peiriods of time. <br />
The differences between our model and the model of Durret and Levin can be seen graphically <br />
in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail conditions <br />
of the model proposed by Durret and Levin (indicated in black font) <br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="3.3.6">3.3.6 The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its <br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in the <br />
production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h4 id="3.3.7">3.3.7 Making it Obvious</h4><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are paths <br />
that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this paths all <br />
have an approximately common origin. In this section we would like to show that the <br />
origin of these paths is practically the same, and that in that sense we have designed <br />
a true randomizer (since, as mentioned before, the imprecisions that result in the <br />
experimental measurements will make it impossible to make the initial <br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three <br />
different strains of E. coli to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<p><br />
We modeled our results using Matlab. <br />
As can be seen in the graphs below, each of the strains can survive if their <br />
initial density in only tree hundredths (a.u.) greater than the other two strains' <br />
initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<h2>References</h2><br />
<p><br />
<ul><br />
<li>Patrick C. Seed et al. "Activation of the Pseudomonas aeruginosa lasI Gene by LasR and the Pseudomonas Autoinducer PAI: an Autoinduction Regulatory Hierarchy" JOURNAL OF BACTERIOLOGY (1995)</li> <br />
<li>Shotaro Ayukawa et al. "Construction of a genetic AND gate under a new standard for assembly of genetic parts" BMC Genomics (2010)</li> <br />
<li>Robert Sidney Cox, III et al. "Programming gene expression with combinatorial promoters" molecular system biology (2007)</li> <br />
<li>Rolf Lutz et al. "Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements" Nucleic Acids Research (1997)</li> <br />
<li>Karina B.Xavier and Bonnie L.Bassler 2004. Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 238-248</li><br />
<li>Ting Xue, Liping Zhao, Haipeng Sun, Xianxuan Zhou, Baolon Sun 2009. LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing. Cell Research (2009), p.1258-1268</li><br />
<li>Liang Wang, Yoshifumi Hashimoto, Chen-Yu Tsao, James J.Valdes, and William E.Bentley 2004. Cyclic AMP(cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 2066-2076</li> <br />
<li>Jean Livet, Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, Robyn A. Bennis, Joshua R. Sanes and Jeff W. Lichtman (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature.</li><br />
<li>Kimi Araki, Masatake Araki1 and Ken-ichi Yamamura (2002). Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites, Nucleic Acids Research</li><br />
<li>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171. </li><br />
<br />
</ul><br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htmTeam:Tokyo Tech/Projects/RPS-game/index.htm2011-10-28T10:13:26Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#1">1. The Hands</a></li><br />
<li><br />
<a href="#2">2. The Judges</a><br />
<ul><br />
<li><a href="#2.1">2.1 Using AND-Gate promoters to create Judges</a></li><br />
<li><a href="#2.2">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</a></li><br />
<li><a href="#2.3">2.3 Improving PlsrA</a></li><br />
<li><a href="#2.4">2.4 Improving Plas</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="#3">3. The Randomizers</a><br />
<ul><br />
<li><a href="#3.1">3.1 Single Colony Isolation</a></li><br />
<li><br />
<a href="#3.2">3.2 Conditional Knockout by Recombination</a><br />
<ul><br />
<li><a href="#3.2.1">3.2.1 The Requirements</a></li><br />
<li><a href="#3.2.2">3.2.2 The Mechanism</a></li><br />
<li><a href="#3.2.3">3.2.3 Testing the Lox Cassettes</a></li><br />
<li><a href="#3.2.4">3.2.4 Playing Fair: Future Work</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="#3.3">3.3 Survival of one strain</a><br />
<ul><br />
<li><a href="#3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</a></li><br />
<li><a href="#3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#3.3.4">3.3.4 The Old Model</a></li><br />
<li><a href="#3.3.5">3.3.5 Our New Model</a></li><br />
<li><a href="#3.3.6">3.3.6 The Biological Meaning of our Model</a></li><br />
<li><a href="#3.3.7">3.3.7 Making it Obvious</a></li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
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<h1> Rock-Paper-Scissors game </h1><br />
<h2> Introduction </h2><br />
<br />
<br />
<div align= "center"><br />
<span class="top"> The Hands </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/1/13/Handzu.png" width="300px" /><br />
</div><br />
<p><br />
The first step towards making an RPS game that can be played <br />
between humans and bacteria is giving each player a set of <br />
signaling molecules through which they can communicate their <br />
choice of rock, paper or scissors. For that purpose we created <br />
two sets of three signaling molecules corresponding each to rock, paper or scissors. <br />
For humans we used IPTG, aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Judges </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" width="350px" /><br />
<table><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" width="360px" /></td><br />
<td><img src="http://partsregistry.org/wiki/images/f/f4/LsrA_promoter_activity_introduction.png" width="400px" /></td><br />
</tr><br />
</table><br />
</div><br />
<br />
<p><br />
Although we defined a set of six signaling molecules that can be used to <br />
play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, we designed a set of <br />
<span class="name">E. coli</span> that act as judges. Because there were no working parts related to AI-2 or 3OC12-HSL, we constructed new working lasI promoter, lsrA promoter and LsrR coding gene. <br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Randomizers </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/7/79/Titech-rps-randomizers.png" width="750px" /><br />
</div><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. <br />
To do so, we designed three kinds of randomizers.<br />
</p><br />
<br />
<p><br />
<h2 id="1">1. The Hands</h2><br />
<img src="https://static.igem.org/mediawiki/2011/4/4b/Image001.png" width="600px" style="float:right;"/><br />
<p><br />
The first step towards making an RPS game that can be played between <br />
humans and bacteria is giving each player a set of signaling molecules <br />
through which they can communicate their choice of rock, paper or scissors. <br />
For that purpose we created two sets of three signaling molecules <br />
corresponding each to rock, paper or scissors. For humans we used IPTG, <br />
aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<br />
<h2 id="2" style="clear:both;">2. The Judges</h2><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" alt="the Judge" style="float: left;" /><br />
<p><br />
Although we defined a set of six signaling molecules that can be used <br />
to play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, <br />
we designed a set of <span class="name">E. coli</span> that act as judges. <br />
Each Judge <span class="name">E. coli</span> has an AND-gate promoter and <br />
a fluorescent protein gene that is expressed when the AND-gate promoter <br />
is activated. In this way, the Judge <span class="name">E. coli</span> can <br />
let us know its decision by producing GFP, RFP or CFP to indicate whether <br />
humans win, lose or it is a tie, respectively.<br />
</p><br />
<br />
<h3 id="2.1" style="clear:both;">2.1 Using AND-Gate promoters to create Judges</h3><br />
<p><br />
The first step to make the Judge <span class="name">E. coli</span> was to <br />
find a logic device which could allow the Judge to decide who the winner of the RPS game was. <br />
We found that the AND-gate promoters would fit perfectly for that purpose, <br />
since they can take two signaling molecules as inputs and produce one indicator <br />
as output. Since each of the players has a set of three different signaling <br />
molecules, we need a set of nine Judges, each of which has an AND-gate promoter <br />
that is activated only by one of the nine possible pairs of signaling molecules. <br />
These combinations are shown in the image below.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Tt-image021.png" width="600px" /><br />
</div><br />
<br />
<p><br />
Our next mission was then to check if there were AND-gate promoters BioBricks <br />
that we could use. We searched in the Registry and found a potential AND-gate <br />
promoter designed by iGEM 2007's team Tokyo_Tech. This potential AND-gate <br />
promoter is designed to be activated by the addition of both IPTG and 3OC6-HSL. <br />
However, there was no data showing the IPTG dependency of this promoter, <br />
so we did experiments and confirmed this dependency for the first time in iGEM. <br />
We concluded that the addition of both IPTG and 3OC6-HSL regulates the activity <br />
of this AND-gate promoter. In this way, we completed the construction of one of <br />
the Judges <span class="name">E. coli</span>, which proves in principle that <br />
our game is feasible. To know the detailed method about this assay, <br />
please see <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.">here</a>.<br />
</p><br />
<br />
<div style="float:left"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/BBa_I751101_graph3.png" width="400px" /><br /><br />
<span class="graph_title">Fig 2.1 Tokyo_Tech AND-gate promoter</span><br />
</div><br />
<p><br />
This Plux-lac hybrid promoter contains two LacI operators, a LuxR operator and luxR. <br />
We introduced this part into LacI expressing <span class="name">E. coli</span> strain. <br />
Because IPTG controls the binding of LacI to two LacI-operator parts and 3OC6-HSL <br />
controls the binding of LuxR to a LuxR-operator part, the <span class="gene">gfp</span> <br />
gene activity of the reporter part is dually regulated by IPTG and 3OC6-HSL. <br />
We used promoterless pSB3K3-<span class="gene">gfp</span> (BBa_J54103) as a negative control, <br />
and pAC-P&lambda;-<span class="gene">gfp</span> (chloramphenicol-resistance), which constitutively expressed GFP, <br />
as a positive control. To know about the mechanism of this promoter click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.0.">here</a>.<br />
</p><br /><br />
<br />
<h3 id="2.2" style="clear:both;">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</h3><br />
<br />
<p><br />
In the process of constructing enough AND-gates that could suffice the needs <br />
of our RPS game design, we discovered two faulty BioBricks: lsrA promoter (BBa_K117002) and las promoter (BBa_J64010). <br />
Because of these faulty parts, the Judge <span class="name">E. coli</span> <br />
set we had designed could only sense the Player <span class="name">E. coli</span>'s <br />
signaling molecule 3OC6-HSL (Rock). This ultimately led to an unfair game <br />
because humans could win every time they played with the Paper signaling molecule <br />
(for more on the &ldquo;Sad story of the Rock Player&rdquo; click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/the_sad_stroy_of_the_rock_player">here</a>).<br />
</p><br />
<p><br />
To fix this problem, we improved the old defective las and lsrA promoters parts <br />
by making new parts that work! As can be seen in the experimental information <br />
below (see “Improving lsrA promoter” and “Improving las promoter”), <br />
we confirmed our lasI promoter (<a href="http://partsregistry.org/Part:BBa_K649000">BBa_K649000</a>) <br />
and lsrA promoter (<a href="http://partsregistry.org/Part:BBa_K649100">BBa_K649100</a>) work perfectly!<br />
</p><br />
<p><br />
Since the lsrA promoter plays a key role in the correct functioning of AI-2, <br />
fixing these parts now allows us to use AI-2 as a signaling molecule, which is a <br />
promising advance because of the characteristics of <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.0.">the AI-2 mechanism</a>.<br />
This mechanism prevents AI-2 from cross-talking with other signaling molecules <br />
such as AHL. Hence, this signaling molecule is a very powerful <br />
tool to build complex Synthetic Biology systems.<br />
</p><br />
<p><br />
Finally, one thing we would like outline is that although the promoters we made <br />
are single input promoters, confirmation of their activity is required as a <br />
reference to construct AND gate promoters. Therefore, we solved important issues <br />
and made significant advances towards constructing AND-gate promoters. This allows <br />
<span class="name">E. coli</span> to also choose the signaling molecules <br />
corresponding to Paper and Scissors, so we have again a working RPS game design.<br />
</p><br />
<br />
<h3 id="2.3">2.3 Improving PlsrA</h3><br />
<br />
<div style="float: left;"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4e/PlsrAactivity.png" alt="activity" width="400px" /><br /><br />
<span class="graph_title">Fig 2.2 Not working lsrA promoter(BBa_K117002) <br /> activity and our new lsrA promoter(BBa_K649100)</span> activity<br />
</div><br /><br />
<p><br />
We confirmed that the lsrA promoter (BBa_K117002) does not work properly <br />
(samples used our experiment are listed in Table 2.1 below). The fluorescence intensity of GFP of lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>) was lower <br />
even than those of the negative control (Fig.2.2), which clearly shows that <br />
lsrA promoter(BBa_K117002) does not work as expected. In this experiment, <br />
we measured transcriptional activity of lsrA promoter by introducing a <br />
<span class="gene">gfp</span> gene downstream of this promoter (Fig.2.3).<br />
</p><br />
<br />
<table style="clear:both;" border="1" align="center"><br />
<caption>Table 2.1 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="4">JD22597</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB1A2</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 (<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a>)</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 ((<a href="http://partsregistry.org/Part:BBa_K117002">BBa_K117002</a>)-<span class="gene">gfp</span>)</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Lasr3.png" alt="Fig2.3" width="200px" /><br /><br />
<span class="graph_title">Fig2.3 lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>)</span><br />
</div><br />
<br />
<p><br />
To solve this problem, we created the first working iGEM lsrA promoter (BBa_K649100). <br />
Its fluorescence intensity was much higher than that from a promoter-less <br />
<span class="gene">gfp</span> negative control plasmid, showing that our new <br />
lsrA promoter works(Fig2.5). In this experiment, we measured the transcriptional <br />
activity of our lsrA promoter by introducing a <span class="gene">gfp</span> gene downstream of <br />
the promoter(BBa_K649104, Fig2.4). Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.">here</a>.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/ff/Lasr4.png" alt="Fig2.4" width="200px"><br /><br />
<span class="graph_title">Fig 2.4 lsrA promoter-<span class="gene">gfp</span>(BBa_K649104)</span><br />
</div><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/LsrR_repression1.png" alt="Fig4" width="600px"><br /><br />
<span class="graph_title">Fig2.5. LsrR represses lsrA promoter.</span><br />
</div><br />
<br />
<p><br />
Moreover, this promoter can be repressed by our new LsrR part(BBa_K649105). <br />
(samples used our experiments are listed in Table 2.2 below) The fluorescence <br />
intensity of GFP of sample 3 was three times as large as <br />
that of sample 4. This result shows that LsrR successfully repressed <br />
lsrA promoter. In this experiment, we measured LsrR repression activity by <br />
introducing a <span class="gene">gfp</span> gene downstream of lsrA promoter (BBa_K649105, Fig.2.6).<br />
Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#6.">here</a>.<br />
</p><br />
<br />
<table align="center" border="1"><br />
<caption>Table2.2 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="2">JM2.300</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td rowspan="2">MG1655</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span>-PlsrR-<span class="gene">lsrR</span> on pSB3K3</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Lasr7.png" alt="Fig.5" width="200px"><br /><br />
<span class="graph_title">Fig2.6 lsrA promoter-<span class="gene">gfp</span>-lsrR promoter-<span class="gene">lsrR</span>(BBa_K649105)</span><br />
</div><br />
<br />
<h3 id="2.4">2.4 Improving las promoter</h3><br />
<br />
<table align="center"> <br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/8/80/BBa_J64010_graph3.png" align="left" height="330px" /><br />
<span class="graph_title">Fig2.7 (a)</span><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" align="right" height="330px" /><br />
<span class="graph_title">(b)</span><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Fig2.7 (a): Not working lasI promoter (BBa_J64010). <br />
Fig2.7 (b): New working lasI promoter (BBa_K649000) we made. <br />
We confirmed it works as expected. In our assay, we used the same asR <br />
regulator part used in the assay of BBa_ J64010. Clearly, for our <br />
part the fluorescence intensity of 3OC12-HSL+ was higher than that of 3OC12-HSL-.<br /><br />
To know detailed methods about these lasI promoter assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#2.">previous part</a> <br />
and <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3.">new part</a>. <br />
</p><br />
<p><br />
To prove that the LasR regulator used in our PlasI assay works, we did another assay. <br />
Details about this assay can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#1.">here.</a><br />
</p><br />
<br />
<h2 id="3">3. The Randomizers</h2><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. To do so, we designed <br />
three kinds of randomizers: one kind which needs of three types of bacteria <br />
(each of which produces one of the three RPS signaling molecules), <br />
and the other kind that needs of only one type of bacteria which can <br />
synthetize each of the three signaling molecules one at a time and randomly. <br />
Namely, the randomizers are Single Colony Isolation, <br />
Survival of one Strain and Conditional Knockout by Recombination.<br />
</p><br />
<br />
<h3 id="3.1">3.1 Single Colony Isolation</h3><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Image041.png" style="float:right;"/><br />
<p><br />
This is our simplest randomizer design. To make sure <span class="name">E. coli</span> <br />
chooses any of its signaling molecules with equal probability, we put the constructs <br />
for each molecule inside a different bacterium, so we create three types of bacteria: <br />
one synthetizing the corresponding signaling molecule for rock, other synthetizing <br />
the corresponding signaling molecule for paper, and lastly one synthetizing the <br />
corresponding signaling molecule for scissors. By randomly isolating a single colony <br />
out of the many colonies that result from the mixing between the three types of <br />
<span class="name">E. coli</span>, we get a random output as <br />
<span class="name">E. coli</span>'s choice for the RPS game.<br />
</p><br />
<br />
<h3 id="3.2" style="clear:both">3.2 Conditional Knockout by Recombination</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/0c/Cre-lox_mechanism_1.png" width="550px" align="center" /><br />
</div><br />
<p><br />
Our second randomizer differs from our other two randomizers in that all the <br />
three signaling molecules are produced one at a time and randomly by only one type <br />
of bacteria. We were inspired by a paper about &ldquo;brainbow&rdquo; research <br />
on mice to create this randomizer (Livet J <i>et al.</i>, 2007), which is based <br />
on the recombination mechanism of the enzyme Cre and the lox sequences. <br />
We designed a Cre-Lox system which allows <span class="name">E. coli</span> <br />
to express one of its three signaling molecules by means of conditional knockout. <br />
The design is depicted in Fig 1. It should be noted this randomizer is designed <br />
to be used at a single-cell level. When this randomizer is used in groups of cells, <br />
the different signals released by the cells will mix. In this case, by using microfluidic <br />
devices or isolating single colony of bacteria, we can obtain only one of the signaling <br />
molecules produced by the initial group of cells.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/3d/Fig_3.1_-_in_one_cell_1.png" width="700px" align="center" /><br />
</div><br />
<div class="graph_title"><br />
Fig 3.1 - (a) Cre recombinase construction. (b) lox cassettes distribution for the randomizer design<br />
</div><br />
<br />
<h4 id="3.2.1">3.2.1 The Requirements</h4><br />
<br />
<p><br />
Basically, each pair of lox sites (indicated by the same color) mark the points <br />
which the enzyme Cre will excise (they will be cut off the backbone along with the <br />
sequence between them). For a design that allows choosing randomly one of <br />
<span class="name">E. coli</span>’s three signaling molecules, at least two <br />
cassettes of lox sites are needed. When these two cassettes of lox sites and <br />
protein coding sequences are arranged as in Fig3.1(b), <br />
only one signaling molecule is produced.<br />
</p><br />
<p><br />
Our design also required a way to control luxI gene's expression. <br />
To do so, we used an inducible promoter instead of a constitutive promoter. <br />
A constitutive promoter would have caused luxI gene to be expressed beforehand and <br />
could lead to an <span class="name">E. coli</span> producing two signaling molecules <br />
at the same time (the equivalent of showing two hands in the RPS game). In contrast, <br />
the inducible promoter prevents a particular gene from expressing preferentially.<br />
</p> <br />
<p><br />
One last but not less important requirement for our randomizer is that it should <br />
express each of the three signals not only one at a time, but also with the same <br />
probability. This makes the game fair in the sense that <br />
<span class="name">E. coli</span>’s choice in the RPS game is not predictable.<br />
</p><br />
<br />
<h4 id="3.2.2">3.2.2 The Mechanism</h4><br />
<br />
<p><br />
When the blue cassette of Lox sites is excised (Fig 3.1), the signaling molecule <br />
coded by lasI (3OC6-HSL) will be produced. Likewise, when the black Lox cassette <br />
is excised, the signaling molecule coded by luxS (AI-2) will be produced. On the other hand, <br />
a third possible outcome is that recombination does not take place. In this case, <br />
the signaling molecule coded by luxI (3OC6-HSL) will be produced. Also note that excision <br />
of one kind of lox cassette removes the remaining cassette, <br />
thereby preventing further recombination.<br />
</p><br />
<p><br />
As mentioned before, one of the requirements for our randomizer was to have at least <br />
two lox cassettes. This prevents excision of lox sites from different cassettes <br />
(for example one blue lox site and one black lox site). Because the lox71-lox66 cassette and the lox2272-lox2272 cassette are incompatible (Zorana Carter and Daniela Delneri <i>et al.</i>, Yeast 2010), we can use them to build our randomizer.<br />
</p><br />
<br />
<h4 id="3.2.3">3.2.3 Testing the Lox Cassettes</h4><br />
<br />
<p><br />
As stated above, we need two lox cassettes of different recombination frequency <br />
for randomizer. Because there was no working lox parts in registry, we constructed <br />
three original BioBricks for testing whether lox2272 and lox71/66 cassettes work. For <br />
the convenience of testing, fluorescence expressing genes were used in place of signal <br />
molecular expressing genes in construction. After figuring out their working <i>in vitro</i>, we tested them <i>in vivo</i> by detecting red and green fluorescence through fluoro imager and flow cytometer. Furthermore, we compared the relative recombination frequency of two cassettes . Our lox Cassettes constructions <br />
were working properly, and their recombination frequency were different from each other.<br />
</p><br />
<br />
<ul><br />
<li><br />
PlacIQ-lox2272-<span class="gene">gfp</span>-lox2272<a href="http://partsregistry.org/Part:BBa_K649200">(BBa_K649200)</a><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/Lox-gfp-lox_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox2272-<span class="gene">rfp</span>-lox2272-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649201">(BBa_K649201)</a><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Lox2272-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox71-<span class="gene">rfp</span>-lox66-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649202">(BBa_K649202)</a><br />
<img src="https://static.igem.org/mediawiki/2011/b/b4/Lox71-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
</ul><br />
<br />
<p><br />
The <i>in vitro</i> assay with K649200 was made in advance. The preliminary experiment allowed <br />
us to confirm that the Cre-mediated recombination on lox2272 cassette works as designed. <br />
In the assay, Cre recombinase was added to the linear DNA and incubated for 0.5, 2, and 4 hours. <br />
Images of the experiments have been added below.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/6/60/In_vitro_lox_gfp_lox.png" style="float: left;"/><br />
<p><br />
When checking the result by electrophoresis, there were several bands in samples to <br />
which Cre was added(1st, 2nd lane from right which corresponds to 4 hr and 2 hr respectively). <br />
It indicates that excision of the lox sites successfully occurred. <br />
To know detailed about this assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#7."><i>in vitro</i> assay for lox2272</a><br />
</p><br />
<br />
<p><br />
For the <i>in vivo</i> assay, by detecting fluorescenc levels of GFP and mCherry, <br />
we could determine whether recombination occured in K649201 and K649202 and compare <br />
relative recombination frequency between two of them. We prepared a competent cell <br />
JM2.300 into which P<sub>BAD</sub>/araC-Cre(pSB1A2, BBa_I718008) had been constructed. <br />
Subsequently, our BioBrick was constructed into the cell.<br />
</p><br />
<br />
<table border="1" align="center"> <br />
<tr><br />
<th colspan="2">sample</th><br />
<th>arabinose</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">-</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br /> : negative control </td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
</table><br />
<br />
<p><br />
The strain was grown in a 3 mL liquid culture, and 75 &micro;L of 2 M arabinose was added <br />
to induce Cre expression. We used two controls for the experiment. One was the same strain <br />
without arabinose induction, and the other was JM2.300 strain which was induced by arabinose <br />
and had only our BioBrick. All the strains were cultured each for periods of 0.5, 1, 2, and 4 hours, <br />
and in each case the florescence levels were measured by flow cytometer and FLA. <br />
To know the detailed method about this assay, please see here <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8."><i>in vivo</i> assay for lox cassettes</a>.<br />
</p><br />
<br />
<br />
<br />
<p><br />
We confirmed our results optically by taking florescence images. <br />
K649201 transformants with with 0.5 hr-induction of Cre in liquid medium and its two control strains were plated and incubated in 37&deg;C for 12 hours. Images of the three conditions were taken using red <br />
florescence filter, green florescence filter and no filter as shown below, respectively.<br />
</p><br />
<br />
<table align="center"><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/1e/Image146.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(a)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/7/76/Image144.png/800px-Image144.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/2/27/Image142.png/800px-Image142.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(c)</td><br />
</tr><br />
</table><br />
<br />
<p><br />
Fig 3.2 Cre-meditated recombination at lox2272 cassette. Cre-induction period of 0.5 hr<br />
(a)Overlay of Green and Red channel. The leftmost is a negative control which don't have Cre-expressing <br />
plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick K649201. <br />
The rightmost is a uninduced strain which has both plasmid like as the center. <br />
(b)Detection of GFP. The order of samples is same as above. <br />
(c)Detection of mCherry. The order of samples is same as above.<br />
</p><br />
<p><br />
On the sample with the P<sub>BAD</sub>/araC-Cre construction, we found that recombination occurred <br />
when arabinose was added. In contrast to this result, when we measured the levels of the sample <br />
without the P<sub>BAD</sub>/araC-Cre construction, we found that the GFP levels were far lower than <br />
those of the sample with the P<sub>BAD</sub>/araC-Cre construction. This clearly proves that our <br />
lox constructions, both in K649201 and K649202, respond correctly to the effects of Cre recombinase. <br />
A slight detection of green florescence in plate absence of P<sub>BAD</sub>/araC-Cre can be explained<br />
that there happened cross-talk to green channel by FMN(Flavin mononucleotide) or expression of GFP according<br />
to malfunction of terminator before <span class="gene">gfp</span>. We could also observe recombination occurred when arabinose was not <br />
added, which can be explained due to a leaking in the P<sub>BAD</sub>/araC promoter.You can find that K649202 also works well from the images of K649202 on<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8.">here</a>.<br />
</p><br />
<p><br />
Furthermore, we could observe that the arabinose(+) sample of K649202 has higher green/red ratio than that of K649201, which implying the frequency of lox 71/66 casette is higher than that of lox 2272.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/86/111001DK_30_eachpair_111003.png" /><br /><br />
<span class="graph_title">Fig 3.3 Image of six samples of K649201 (up) and K649202 (down) at period of 0.5 hr</span><br />
</div><br />
<br />
<table border="0"> <br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/11/Proportion_2272_0.5hr.png" /></td><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/bb/Proportion_7166_0.5hr.png" /></td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(a)</td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/archive/5/52/20111004160721%21Proportion_area.png" /></td><br />
<td><br />
Fig 3.4 identical plates with Fig 3.3<br /><br />
(a)expression levels of red and green florescence of K649201<br /><br />
(b)expression levels of red and green florescence of K649202<br /><br />
(c)examined area for comparing between red and green florescence at each plate<br /><br />
</td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(c)</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
<p><br />
As we examining green florescence in comparison to red florescence, green expression level was lower than red in <br />
K649201(Fig 3.4(a)), which meaning that considerable plasmids in those cells yet. In contrast, green expression level exceeded red in K649202(Fig 3.4(b)). This result implies that recombination frequency of <br />
lox71/66 cassette is relatively high than that of lox2272 cassette.<br /><br />
<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a2/Flow_cytometer.png"/ width="500px" ></center><br><br />
<p> <span class="graph_title">Fig 3.5 Green fluorescence level of each cell was detected by flow cytometer.<br><br />
(a)arabinose induced strain containing only K649201 and cre-expressing plasmid (b)arabinose supplied strain containing only K649201 (c)arabinose induced starin containing K649202 and cre-expressing plasmid (d)arabinose supplied strain containing only K649202<br><br />
</p><br />
<p><br />
The higher recombination efficiency of lox 71/66 compare to lox2272 was confirmed also by flow cytometer intensity of lox71/66 was higher than that of lox2272, which supports the result detected by FLA. <br />
</span><br />
</p><br />
<br />
<h4 id="3.2.4">3.2.4 Playing Fair: Future Work</h4><br />
<br />
<p><br />
To make each of the outcomes (R, P, and S) equally probable, we are going to quantify the recombination frequency of each lox cassette. This information and adequate Cre induction will be likely to allow us to have an RPS player <br />
<span class="name">E. coli</span> whose choice of either of rock, paper or scissors cannot be predicted.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e6/Regulating_time_1.png" width="700px"/><br />
</div><br />
<br />
<p><br />
In our next experiments, we are going to vary the reaction time and the distance between the lox sites <br />
of each cassette. We believe precise modification of this two parameters must lead to our goal of <br />
making a randomizer in which each of the signaling molecules can be expressed with the same frequency <br />
(which results in each of the outcomes being expressed with the same probability).<br />
</p><br />
<br />
<h3 id="3.3">3.3 Survival of One strain</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" /><br />
</div><br />
<br />
<h4 id="3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</h4><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a randomizer <br />
that can be used in our Rock-Paper-Scissors game, due to the fact that only one of the rival strains <br />
will survive. More specifically, we assign to each of the three rival strains either of Rock, Paper or Scissors, <br />
make them compete for survival and take the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h4 id="3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</h4><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in <br />
1996 by Durret and Levin. In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. <br />
However, the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive <br />
(although it can dominate the system, i.e. have the highest population density, for definite periods of time). <br />
We will discuss more on the limitations we found in this model to be adopted as a <br />
randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h4 id="3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</h4><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to outcompete their rivals: <br />
the production of a toxin (a bacteriocin called colicin) that is toxic to other strains, <br />
resistance to the toxin produced by other strains, and a higher birth rate than their rival strains. <br />
Namely, the three types of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) <br />
and colicin-sensitive <span class="name">E. coli</span> (S). <br />
The colicin-producer outcompetes the colicin-sensitive by producing the colicin. The colicin-sensitive <br />
bacteria outcompetes the colicin-resistant because its birth rate is higher than that of the colicin-resistant. <br />
The colicin-resistant outcompetes the colicin producer because its birth rate is higher <br />
than that of the colicin producer. The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<h4 id="3.3.4">3.3.4 The Old Model</h4><br />
<br />
<p><br />
In the model described by Durret and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). <br />
However, as will be explained afterwards, this results in a loss of <br />
balance that does not allow building a true randomizing system.<br />
</p><br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durret and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
<br />
</div><br />
<br />
<h4 id="3.3.5">3.3.5 Our New Model</h4><br />
<p><br />
As mentioned before, the model proposed by Durret and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it <br />
a biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive <br />
by outcompeting the other two strains, which will die. More specifically, we limited the resistance of the <br />
colicin-resistant bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and <br />
to the sensitive strain, and additionally the resistant strain would also be vulnerable to the colicin produced by <br />
the colicin-producer. Since which strain will be the one that survives is determined by very small differences in <br />
the initial concentrations of the three different populations of bacteria, in practice this systems becomes a <br />
randomizer because of the imprecisions in the measurements that result, for example, when using micropipettes. <br />
This randomizer describes a new competition dynamic that could not be reproduced in the previous model proposed <br />
by Durret and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p><br />
If we set the parameters as follows<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, <br />
we get a graph which clearly shows there are stable points on each of the three axes <br />
(Figure 1, Up).<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
</div><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations <br />
we have set all of the three strains may ultimately survive for infinite peiriods of time. <br />
The differences between our model and the model of Durret and Levin can be seen graphically <br />
in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail conditions <br />
of the model proposed by Durret and Levin (indicated in black font) <br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="3.3.6">3.3.6 The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its <br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in the <br />
production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h4 id="3.3.7">3.3.7 Making it Obvious</h4><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are paths <br />
that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this paths all <br />
have an approximately common origin. In this section we would like to show that the <br />
origin of these paths is practically the same, and that in that sense we have designed <br />
a true randomizer (since, as mentioned before, the imprecisions that result in the <br />
experimental measurements will make it impossible to make the initial <br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three <br />
different strains of E. coli to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<p><br />
We modeled our results using Matlab. <br />
As can be seen in the graphs below, each of the strains can survive if their <br />
initial density in only tree hundredths (a.u.) greater than the other two strains' <br />
initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<h2>References</h2><br />
<p><br />
<ul><br />
<li>Patrick C. Seed et al. "Activation of the Pseudomonas aeruginosa lasI Gene by LasR and the Pseudomonas Autoinducer PAI: an Autoinduction Regulatory Hierarchy" JOURNAL OF BACTERIOLOGY (1995)</li> <br />
<li>Shotaro Ayukawa et al. "Construction of a genetic AND gate under a new standard for assembly of genetic parts" BMC Genomics (2010)</li> <br />
<li>Robert Sidney Cox, III et al. "Programming gene expression with combinatorial promoters" molecular system biology (2007)</li> <br />
<li>Rolf Lutz et al. "Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements" Nucleic Acids Research (1997)</li> <br />
<li>Karina B.Xavier and Bonnie L.Bassler 2004. Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 238-248</li><br />
<li>Ting Xue, Liping Zhao, Haipeng Sun, Xianxuan Zhou, Baolon Sun 2009. LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing. Cell Research (2009), p.1258-1268</li><br />
<li>Liang Wang, Yoshifumi Hashimoto, Chen-Yu Tsao, James J.Valdes, and William E.Bentley 2004. Cyclic AMP(cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 2066-2076</li> <br />
<li>Jean Livet, Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, Robyn A. Bennis, Joshua R. Sanes and Jeff W. Lichtman (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature.</li><br />
<li>Kimi Araki, Masatake Araki1 and Ken-ichi Yamamura (2002). Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites, Nucleic Acids Research</li><br />
<li>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171. </li><br />
<br />
</ul><br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htmTeam:Tokyo Tech/Projects/RPS-game/index.htm2011-10-28T10:10:36Z<p>Takuya 1613: </p>
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<ul><br />
<li><a href="#1">1. The Hands</a></li><br />
<li><br />
<a href="#2">2. The Judges</a><br />
<ul><br />
<li><a href="#2.1">2.1 Using AND-Gate promoters to create Judges</a></li><br />
<li><a href="#2.2">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</a></li><br />
<li><a href="#2.3">2.3 Improving PlsrA</a></li><br />
<li><a href="#2.4">2.4 Improving Plas</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="#3">3. The Randomizers</a><br />
<ul><br />
<li><a href="#3.1">3.1 Single Colony Isolation</a></li><br />
<li><br />
<a href="#3.2">3.2 Conditional Knockout by Recombination</a><br />
<ul><br />
<li><a href="#3.2.1">3.2.1 The Requirements</a></li><br />
<li><a href="#3.2.2">3.2.2 The Mechanism</a></li><br />
<li><a href="#3.2.3">3.2.3 Testing the Lox Cassettes</a></li><br />
<li><a href="#3.2.4">3.2.4 Playing Fair: Future Work</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="#3.3">3.3 Survival of one strain</a><br />
<ul><br />
<li><a href="#3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</a></li><br />
<li><a href="#3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</a></li><br />
<li><a href="#3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</a></li><br />
<li><a href="#3.3.4">3.3.4 The Old Model</a></li><br />
<li><a href="#3.3.5">3.3.5 Our New Model</a></li><br />
<li><a href="#3.3.6">3.3.6 The Biological Meaning of our Model</a></li><br />
<li><a href="#3.3.7">3.3.7 Making it Obvious</a></li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
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<h1> Rock-Paper-Scissors game </h1><br />
<h2> Introduction </h2><br />
<br />
<br />
<div align= "center"><br />
<span class="top"> The Hands </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/1/13/Handzu.png" width="300px" /><br />
</div><br />
<p><br />
The first step towards making an RPS game that can be played <br />
between humans and bacteria is giving each player a set of <br />
signaling molecules through which they can communicate their <br />
choice of rock, paper or scissors. For that purpose we created <br />
two sets of three signaling molecules corresponding each to rock, paper or scissors. <br />
For humans we used IPTG, aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Judges </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" width="350px" /><br />
<table><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" width="360px" /></td><br />
<td><img src="http://partsregistry.org/wiki/images/f/f4/LsrA_promoter_activity_introduction.png" width="400px" /></td><br />
</tr><br />
</table><br />
</div><br />
<br />
<p><br />
Although we defined a set of six signaling molecules that can be used to <br />
play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, we designed a set of <br />
<span class="name">E. coli</span> that act as judges. Because there were no working parts related to AI-2 or 3OC12-HSL, we constructed new working lasI promoter, lsrA promoter and LsrR coding gene. <br />
</p><br />
<hr /><br />
<br />
<div align= "center"><br />
<span class="top"> The Randomizers </span><br /><br />
<img src="https://static.igem.org/mediawiki/2011/7/79/Titech-rps-randomizers.png" width="750px" /><br />
</div><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. <br />
To do so, we designed three kinds of randomizers.<br />
</p><br />
<br />
<p><br />
<h2 id="1">1. The Hands</h2><br />
<img src="https://static.igem.org/mediawiki/2011/4/4b/Image001.png" width="600px" style="float:right;"/><br />
<p><br />
The first step towards making an RPS game that can be played between <br />
humans and bacteria is giving each player a set of signaling molecules <br />
through which they can communicate their choice of rock, paper or scissors. <br />
For that purpose we created two sets of three signaling molecules <br />
corresponding each to rock, paper or scissors. For humans we used IPTG, <br />
aTc and salicylate, respectively. <br />
For <span class="name">E. coli</span> we used 3OC6-HSL, 3OC12-HSL and AI-2, respectively.<br />
</p><br />
<br />
<h2 id="2" style="clear:both;">2. The Judges</h2><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Judge.png" alt="the Judge" style="float: left;" /><br />
<p><br />
Although we defined a set of six signaling molecules that can be used <br />
to play the RPS game, we still need to find a way to know who wins the game. <br />
To know who the winner of each game is, <br />
we designed a set of <span class="name">E. coli</span> that act as judges. <br />
Each Judge <span class="name">E. coli</span> has an AND-gate promoter and <br />
a fluorescent protein gene that is expressed when the AND-gate promoter <br />
is activated. In this way, the Judge <span class="name">E. coli</span> can <br />
let us know its decision by producing GFP, RFP or CFP to indicate whether <br />
humans win, lose or it is a tie, respectively.<br />
</p><br />
<br />
<h3 id="2.1" style="clear:both;">2.1 Using AND-Gate promoters to create Judges</h3><br />
<p><br />
The first step to make the Judge <span class="name">E. coli</span> was to <br />
find a logic device which could allow the Judge to decide who the winner of the RPS game was. <br />
We found that the AND-gate promoters would fit perfectly for that purpose, <br />
since they can take two signaling molecules as inputs and produce one indicator <br />
as output. Since each of the players has a set of three different signaling <br />
molecules, we need a set of nine Judges, each of which has an AND-gate promoter <br />
that is activated only by one of the nine possible pairs of signaling molecules. <br />
These combinations are shown in the image below.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Tt-image021.png" width="600px" /><br />
</div><br />
<br />
<p><br />
Our next mission was then to check if there were AND-gate promoters BioBricks <br />
that we could use. We searched in the Registry and found a potential AND-gate <br />
promoter designed by iGEM 2007's team Tokyo_Tech. This potential AND-gate <br />
promoter is designed to be activated by the addition of both IPTG and 3OC6-HSL. <br />
However, there was no data showing the IPTG dependency of this promoter, <br />
so we did experiments and confirmed this dependency for the first time in iGEM. <br />
We concluded that the addition of both IPTG and 3OC6-HSL regulates the activity <br />
of this AND-gate promoter. In this way, we completed the construction of one of <br />
the Judges <span class="name">E. coli</span>, which proves in principle that <br />
our game is feasible. To know the detailed method about this assay, <br />
please see <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.">here</a>.<br />
</p><br />
<br />
<div style="float:left"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/BBa_I751101_graph3.png" width="400px" /><br /><br />
<span class="graph_title">Fig 2.1 Tokyo_Tech AND-gate promoter</span><br />
</div><br />
<p><br />
This Plux-lac hybrid promoter contains two LacI operators, a LuxR operator and luxR. <br />
We introduced this part into LacI expressing <span class="name">E. coli</span> strain. <br />
Because IPTG controls the binding of LacI to two LacI-operator parts and 3OC6-HSL <br />
controls the binding of LuxR to a LuxR-operator part, the <span class="gene">gfp</span> <br />
gene activity of the reporter part is dually regulated by IPTG and 3OC6-HSL. <br />
We used promoterless pSB3K3-<span class="gene">gfp</span> (BBa_J54103) as a negative control, <br />
and pAC-P&lambda;-<span class="gene">gfp</span> (chloramphenicol-resistance), which constitutively expressed GFP, <br />
as a positive control. To know about the mechanism of this promoter click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#4.0.">here</a>.<br />
</p><br /><br />
<br />
<h3 id="2.2" style="clear:both;">2.2 Creating Parts that responded correctly to our set of Signaling Molecules</h3><br />
<br />
<p><br />
In the process of constructing enough AND-gates that could suffice the needs <br />
of our RPS game design, we discovered two faulty BioBricks: lsrA promoter (BBa_K117002) and las promoter (BBa_J64010). <br />
Because of these faulty parts, the Judge <span class="name">E. coli</span> <br />
set we had designed could only sense the Player <span class="name">E. coli</span>'s <br />
signaling molecule 3OC6-HSL (Rock). This ultimately led to an unfair game <br />
because humans could win every time they played with the Paper signaling molecule <br />
(for more on the &ldquo;Sad story of the Rock Player&rdquo; click <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/the_sad_stroy_of_the_rock_player">here</a>).<br />
</p><br />
<p><br />
To fix this problem, we improved the old defective las and lsrA promoters parts <br />
by making new parts that work! As can be seen in the experimental information <br />
below (see “Improving lsrA promoter” and “Improving las promoter”), <br />
we confirmed our lasI promoter (<a href="http://partsregistry.org/Part:BBa_K649000">BBa_K649000</a>) <br />
and lsrA promoter (<a href="http://partsregistry.org/Part:BBa_K649100">BBa_K649100</a>) work perfectly!<br />
</p><br />
<p><br />
Since the lsrA promoter plays a key role in the correct functioning of AI-2, <br />
fixing these parts now allows us to use AI-2 as a signaling molecule, which is a <br />
promising advance because of the characteristics of <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.0.">the AI-2 mechanism</a>.<br />
This mechanism prevents AI-2 from cross-talking with other signaling molecules <br />
such as AHL. Hence, this signaling molecule is a very powerful <br />
tool to build complex Synthetic Biology systems.<br />
</p><br />
<p><br />
Finally, one thing we would like outline is that although the promoters we made <br />
are single input promoters, confirmation of their activity is required as a <br />
reference to construct AND gate promoters. Therefore, we solved important issues <br />
and made significant advances towards constructing AND-gate promoters. This allows <br />
<span class="name">E. coli</span> to also choose the signaling molecules <br />
corresponding to Paper and Scissors, so we have again a working RPS game design.<br />
</p><br />
<br />
<h3 id="2.3">2.3 Improving PlsrA</h3><br />
<br />
<div style="float: left;"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4e/PlsrAactivity.png" alt="activity" width="400px" /><br /><br />
<span class="graph_title">Fig 2.2 Not working lsrA promoter(BBa_K117002) <br /> activity and our new lsrA promoter(BBa_K649100)</span> activity<br />
</div><br />
<p><br />
We confirmed that the lsrA promoter (BBa_K117002) does not work properly <br />
(samples used our experiment are listed in Table 2.1 below). The fluorescence intensity of GFP of lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>) was lower <br />
even than those of the negative control (Fig.2.2), which clearly shows that <br />
lsrA promoter(BBa_K117002) does not work as expected. In this experiment, <br />
we measured transcriptional activity of lsrA promoter by introducing a <br />
<span class="gene">gfp</span> gene downstream of this promoter (Fig.2.3).<br />
</p><br />
<br />
<table style="clear:both;" border="1" align="center"><br />
<caption>Table 2.1 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="4">JD22597</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB1A2</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 (<a href="http://partsregistry.org/Part:BBa_K649104">BBa_K649104</a>)</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB1A2 ((<a href="http://partsregistry.org/Part:BBa_K117002">BBa_K117002</a>)-<span class="gene">gfp</span>)</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Lasr3.png" alt="Fig2.3" width="200px" /><br /><br />
<span class="graph_title">Fig2.3 lsrA promoter-<span class="gene">gfp</span>((BBa_K117002)-<span class="gene">gfp</span>)</span><br />
</div><br />
<br />
<p><br />
To solve this problem, we created the first working iGEM lsrA promoter (BBa_K649100). <br />
Its fluorescence intensity was much higher than that from a promoter-less <br />
<span class="gene">gfp</span> negative control plasmid, showing that our new <br />
lsrA promoter works(Fig2.5). In this experiment, we measured the transcriptional <br />
activity of our lsrA promoter by introducing a <span class="gene">gfp</span> gene downstream of <br />
the promoter(BBa_K649104, Fig2.4). Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5.">here</a>.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/ff/Lasr4.png" alt="Fig2.4" width="200px"><br /><br />
<span class="graph_title">Fig 2.4 lsrA promoter-<span class="gene">gfp</span>(BBa_K649104)</span><br />
</div><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/LsrR_repression1.png" alt="Fig4" width="600px"><br /><br />
<span class="graph_title">Fig2.5. LsrR represses lsrA promoter.</span><br />
</div><br />
<br />
<p><br />
Moreover, this promoter can be repressed by our new LsrR part(BBa_K649105). <br />
(samples used our experiments are listed in Table 2.2 below) The fluorescence <br />
intensity of GFP of sample 3 was three times as large as <br />
that of sample 4. This result shows that LsrR successfully repressed <br />
lsrA promoter. In this experiment, we measured LsrR repression activity by <br />
introducing a <span class="gene">gfp</span> gene downstream of lsrA promoter (BBa_K649105, Fig.2.6).<br />
Details about this experiment can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#6.">here</a>.<br />
</p><br />
<br />
<table align="center" border="1"><br />
<caption>Table2.2 Samples used our experiment</caption><br />
<tr><br />
<th>Name</th><br />
<th>Strain</th><br />
<th>Plasmid</th><br />
</tr><br />
<tr><br />
<td>sample1</td><br />
<td rowspan="2">JM2.300</td><br />
<td>Ptet-<span class="gene">gfp</span> on pSB6A1</td><br />
</tr><br />
<tr><br />
<td>sample2</td><br />
<td>Promoterless-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample3</td><br />
<td rowspan="2">MG1655</td><br />
<td>PlsrA-<span class="gene">gfp</span> on pSB3K3</td><br />
</tr><br />
<tr><br />
<td>sample4</td><br />
<td>PlsrA-<span class="gene">gfp</span>-PlsrR-<span class="gene">lsrR</span> on pSB3K3</td><br />
</tr><br />
</table><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Lasr7.png" alt="Fig.5" width="200px"><br /><br />
<span class="graph_title">Fig2.6 lsrA promoter-<span class="gene">gfp</span>-lsrR promoter-<span class="gene">lsrR</span>(BBa_K649105)</span><br />
</div><br />
<br />
<h3 id="2.4">2.4 Improving las promoter</h3><br />
<br />
<table align="center"> <br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/8/80/BBa_J64010_graph3.png" align="left" height="330px" /><br />
<span class="graph_title">Fig2.7 (a)</span><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/2/24/BBa_K649001_graph3.png" align="right" height="330px" /><br />
<span class="graph_title">(b)</span><br />
</td><br />
</tr><br />
</table><br />
<p><br />
Fig2.7 (a): Not working lasI promoter (BBa_J64010). <br />
Fig2.7 (b): New working lasI promoter (BBa_K649000) we made. <br />
We confirmed it works as expected. In our assay, we used the same asR <br />
regulator part used in the assay of BBa_ J64010. Clearly, for our <br />
part the fluorescence intensity of 3OC12-HSL+ was higher than that of 3OC12-HSL-.<br /><br />
To know detailed methods about these lasI promoter assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#2.">previous part</a> <br />
and <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#3.">new part</a>. <br />
</p><br />
<p><br />
To prove that the LasR regulator used in our PlasI assay works, we did another assay. <br />
Details about this assay can be found <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#1.">here.</a><br />
</p><br />
<br />
<h2 id="3">3. The Randomizers</h2><br />
<br />
<p><br />
Although our set of six signaling molecules allows us to play RPS with <br />
<span class="name">E. coli</span>, we must make sure <span class="name">E. coli</span> <br />
can choose any of its three signaling molecules with the same probability <br />
in order to be able to play RPS fairly and properly. To do so, we designed <br />
three kinds of randomizers: one kind which needs of three types of bacteria <br />
(each of which produces one of the three RPS signaling molecules), <br />
and the other kind that needs of only one type of bacteria which can <br />
synthetize each of the three signaling molecules one at a time and randomly. <br />
Namely, the randomizers are Single Colony Isolation, <br />
Survival of one Strain and Conditional Knockout by Recombination.<br />
</p><br />
<br />
<h3 id="3.1">3.1 Single Colony Isolation</h3><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/d/d6/Image041.png" style="float:right;"/><br />
<p><br />
This is our simplest randomizer design. To make sure <span class="name">E. coli</span> <br />
chooses any of its signaling molecules with equal probability, we put the constructs <br />
for each molecule inside a different bacterium, so we create three types of bacteria: <br />
one synthetizing the corresponding signaling molecule for rock, other synthetizing <br />
the corresponding signaling molecule for paper, and lastly one synthetizing the <br />
corresponding signaling molecule for scissors. By randomly isolating a single colony <br />
out of the many colonies that result from the mixing between the three types of <br />
<span class="name">E. coli</span>, we get a random output as <br />
<span class="name">E. coli</span>'s choice for the RPS game.<br />
</p><br />
<br />
<h3 id="3.2" style="clear:both">3.2 Conditional Knockout by Recombination</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/0c/Cre-lox_mechanism_1.png" width="550px" align="center" /><br />
</div><br />
<p><br />
Our second randomizer differs from our other two randomizers in that all the <br />
three signaling molecules are produced one at a time and randomly by only one type <br />
of bacteria. We were inspired by a paper about &ldquo;brainbow&rdquo; research <br />
on mice to create this randomizer (Livet J <i>et al.</i>, 2007), which is based <br />
on the recombination mechanism of the enzyme Cre and the lox sequences. <br />
We designed a Cre-Lox system which allows <span class="name">E. coli</span> <br />
to express one of its three signaling molecules by means of conditional knockout. <br />
The design is depicted in Fig 1. It should be noted this randomizer is designed <br />
to be used at a single-cell level. When this randomizer is used in groups of cells, <br />
the different signals released by the cells will mix. In this case, by using microfluidic <br />
devices or isolating single colony of bacteria, we can obtain only one of the signaling <br />
molecules produced by the initial group of cells.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/3/3d/Fig_3.1_-_in_one_cell_1.png" width="700px" align="center" /><br />
</div><br />
<div class="graph_title"><br />
Fig 3.1 - (a) Cre recombinase construction. (b) lox cassettes distribution for the randomizer design<br />
</div><br />
<br />
<h4 id="3.2.1">3.2.1 The Requirements</h4><br />
<br />
<p><br />
Basically, each pair of lox sites (indicated by the same color) mark the points <br />
which the enzyme Cre will excise (they will be cut off the backbone along with the <br />
sequence between them). For a design that allows choosing randomly one of <br />
<span class="name">E. coli</span>’s three signaling molecules, at least two <br />
cassettes of lox sites are needed. When these two cassettes of lox sites and <br />
protein coding sequences are arranged as in Fig3.1(b), <br />
only one signaling molecule is produced.<br />
</p><br />
<p><br />
Our design also required a way to control luxI gene's expression. <br />
To do so, we used an inducible promoter instead of a constitutive promoter. <br />
A constitutive promoter would have caused luxI gene to be expressed beforehand and <br />
could lead to an <span class="name">E. coli</span> producing two signaling molecules <br />
at the same time (the equivalent of showing two hands in the RPS game). In contrast, <br />
the inducible promoter prevents a particular gene from expressing preferentially.<br />
</p> <br />
<p><br />
One last but not less important requirement for our randomizer is that it should <br />
express each of the three signals not only one at a time, but also with the same <br />
probability. This makes the game fair in the sense that <br />
<span class="name">E. coli</span>’s choice in the RPS game is not predictable.<br />
</p><br />
<br />
<h4 id="3.2.2">3.2.2 The Mechanism</h4><br />
<br />
<p><br />
When the blue cassette of Lox sites is excised (Fig 3.1), the signaling molecule <br />
coded by lasI (3OC6-HSL) will be produced. Likewise, when the black Lox cassette <br />
is excised, the signaling molecule coded by luxS (AI-2) will be produced. On the other hand, <br />
a third possible outcome is that recombination does not take place. In this case, <br />
the signaling molecule coded by luxI (3OC6-HSL) will be produced. Also note that excision <br />
of one kind of lox cassette removes the remaining cassette, <br />
thereby preventing further recombination.<br />
</p><br />
<p><br />
As mentioned before, one of the requirements for our randomizer was to have at least <br />
two lox cassettes. This prevents excision of lox sites from different cassettes <br />
(for example one blue lox site and one black lox site). Because the lox71-lox66 cassette and the lox2272-lox2272 cassette are incompatible (Zorana Carter and Daniela Delneri <i>et al.</i>, Yeast 2010), we can use them to build our randomizer.<br />
</p><br />
<br />
<h4 id="3.2.3">3.2.3 Testing the Lox Cassettes</h4><br />
<br />
<p><br />
As stated above, we need two lox cassettes of different recombination frequency <br />
for randomizer. Because there was no working lox parts in registry, we constructed <br />
three original BioBricks for testing whether lox2272 and lox71/66 cassettes work. For <br />
the convenience of testing, fluorescence expressing genes were used in place of signal <br />
molecular expressing genes in construction. After figuring out their working <i>in vitro</i>, we tested them <i>in vivo</i> by detecting red and green fluorescence through fluoro imager and flow cytometer. Furthermore, we compared the relative recombination frequency of two cassettes . Our lox Cassettes constructions <br />
were working properly, and their recombination frequency were different from each other.<br />
</p><br />
<br />
<ul><br />
<li><br />
PlacIQ-lox2272-<span class="gene">gfp</span>-lox2272<a href="http://partsregistry.org/Part:BBa_K649200">(BBa_K649200)</a><br />
<img src="https://static.igem.org/mediawiki/2011/6/6d/Lox-gfp-lox_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox2272-<span class="gene">rfp</span>-lox2272-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649201">(BBa_K649201)</a><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Lox2272-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
<li><br />
PlacIQ-lox71-<span class="gene">rfp</span>-lox66-<span class="gene">gfp</span><a href="http://partsregistry.org/Part:BBa_K649202">(BBa_K649202)</a><br />
<img src="https://static.igem.org/mediawiki/2011/b/b4/Lox71-rfp-lox-gfp_1.png" width="550px"/><br />
</li><br />
</ul><br />
<br />
<p><br />
The <i>in vitro</i> assay with K649200 was made in advance. The preliminary experiment allowed <br />
us to confirm that the Cre-mediated recombination on lox2272 cassette works as designed. <br />
In the assay, Cre recombinase was added to the linear DNA and incubated for 0.5, 2, and 4 hours. <br />
Images of the experiments have been added below.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/6/60/In_vitro_lox_gfp_lox.png" style="float: left;"/><br />
<p><br />
When checking the result by electrophoresis, there were several bands in samples to <br />
which Cre was added(1st, 2nd lane from right which corresponds to 4 hr and 2 hr respectively). <br />
It indicates that excision of the lox sites successfully occurred. <br />
To know detailed about this assay, please see here, <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#7."><i>in vitro</i> assay for lox2272</a><br />
</p><br />
<br />
<p><br />
For the <i>in vivo</i> assay, by detecting fluorescenc levels of GFP and mCherry, <br />
we could determine whether recombination occured in K649201 and K649202 and compare <br />
relative recombination frequency between two of them. We prepared a competent cell <br />
JM2.300 into which P<sub>BAD</sub>/araC-Cre(pSB1A2, BBa_I718008) had been constructed. <br />
Subsequently, our BioBrick was constructed into the cell.<br />
</p><br />
<br />
<table border="1" align="center"> <br />
<tr><br />
<th colspan="2">sample</th><br />
<th>arabinose</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br />P<sub>BAD</sub>/araC-Cre(pSB1A2)</td><br />
<td style="text-align:center;">-</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>PlacIQ-lox-<span class="gene">rfp</span>-lox-<span class="gene">gfp</span>(pSB3K3)<br /> : negative control </td><br />
<td style="text-align:center;">+</td><br />
</tr><br />
</table><br />
<br />
<p><br />
The strain was grown in a 3 mL liquid culture, and 75 &micro;L of 2 M arabinose was added <br />
to induce Cre expression. We used two controls for the experiment. One was the same strain <br />
without arabinose induction, and the other was JM2.300 strain which was induced by arabinose <br />
and had only our BioBrick. All the strains were cultured each for periods of 0.5, 1, 2, and 4 hours, <br />
and in each case the florescence levels were measured by flow cytometer and FLA. <br />
To know the detailed method about this assay, please see here <br />
<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8."><i>in vivo</i> assay for lox cassettes</a>.<br />
</p><br />
<br />
<br />
<br />
<p><br />
We confirmed our results optically by taking florescence images. <br />
K649201 transformants with with 0.5 hr-induction of Cre in liquid medium and its two control strains were plated and incubated in 37&deg;C for 12 hours. Images of the three conditions were taken using red <br />
florescence filter, green florescence filter and no filter as shown below, respectively.<br />
</p><br />
<br />
<table align="center"><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/1e/Image146.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(a)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/7/76/Image144.png/800px-Image144.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/thumb/2/27/Image142.png/800px-Image142.png" style="margin-top:10px;margin-bottom:10px;" width="500px" /></td><br />
<td style="text-align:center;">(c)</td><br />
</tr><br />
</table><br />
<br />
<p><br />
Fig 3.2 Cre-meditated recombination at lox2272 cassette. Cre-induction period of 0.5 hr<br />
(a)Overlay of Green and Red channel. The leftmost is a negative control which don't have Cre-expressing <br />
plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick K649201. <br />
The rightmost is a uninduced strain which has both plasmid like as the center. <br />
(b)Detection of GFP. The order of samples is same as above. <br />
(c)Detection of mCherry. The order of samples is same as above.<br />
</p><br />
<p><br />
On the sample with the P<sub>BAD</sub>/araC-Cre construction, we found that recombination occurred <br />
when arabinose was added. In contrast to this result, when we measured the levels of the sample <br />
without the P<sub>BAD</sub>/araC-Cre construction, we found that the GFP levels were far lower than <br />
those of the sample with the P<sub>BAD</sub>/araC-Cre construction. This clearly proves that our <br />
lox constructions, both in K649201 and K649202, respond correctly to the effects of Cre recombinase. <br />
A slight detection of green florescence in plate absence of P<sub>BAD</sub>/araC-Cre can be explained<br />
that there happened cross-talk to green channel by FMN(Flavin mononucleotide) or expression of GFP according<br />
to malfunction of terminator before <span class="gene">gfp</span>. We could also observe recombination occurred when arabinose was not <br />
added, which can be explained due to a leaking in the P<sub>BAD</sub>/araC promoter.You can find that K649202 also works well from the images of K649202 on<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8.">here</a>.<br />
</p><br />
<p><br />
Furthermore, we could observe that the arabinose(+) sample of K649202 has higher green/red ratio than that of K649201, which implying the frequency of lox 71/66 casette is higher than that of lox 2272.<br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/8/86/111001DK_30_eachpair_111003.png" /><br /><br />
<span class="graph_title">Fig 3.3 Image of six samples of K649201 (up) and K649202 (down) at period of 0.5 hr</span><br />
</div><br />
<br />
<table border="0"> <br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/1/11/Proportion_2272_0.5hr.png" /></td><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/bb/Proportion_7166_0.5hr.png" /></td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(a)</td><br />
<td style="text-align:center;">(b)</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/archive/5/52/20111004160721%21Proportion_area.png" /></td><br />
<td><br />
Fig 3.4 identical plates with Fig 3.3<br /><br />
(a)expression levels of red and green florescence of K649201<br /><br />
(b)expression levels of red and green florescence of K649202<br /><br />
(c)examined area for comparing between red and green florescence at each plate<br /><br />
</td><br />
</tr><br />
<tr><br />
<td style="text-align:center;">(c)</td><br />
<td> </td><br />
</tr><br />
</table><br />
<br />
<p><br />
As we examining green florescence in comparison to red florescence, green expression level was lower than red in <br />
K649201(Fig 3.4(a)), which meaning that considerable plasmids in those cells yet. In contrast, green expression level exceeded red in K649202(Fig 3.4(b)). This result implies that recombination frequency of <br />
lox71/66 cassette is relatively high than that of lox2272 cassette.<br /><br />
<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a2/Flow_cytometer.png"/ width="500px" ></center><br><br />
<p> <span class="graph_title">Fig 3.5 Green fluorescence level of each cell was detected by flow cytometer.<br><br />
(a)arabinose induced strain containing only K649201 and cre-expressing plasmid (b)arabinose supplied strain containing only K649201 (c)arabinose induced starin containing K649202 and cre-expressing plasmid (d)arabinose supplied strain containing only K649202<br><br />
</p><br />
<p><br />
The higher recombination efficiency of lox 71/66 compare to lox2272 was confirmed also by flow cytometer intensity of lox71/66 was higher than that of lox2272, which supports the result detected by FLA. <br />
</span><br />
</p><br />
<br />
<h4 id="3.2.4">3.2.4 Playing Fair: Future Work</h4><br />
<br />
<p><br />
To make each of the outcomes (R, P, and S) equally probable, we are going to quantify the recombination frequency of each lox cassette. This information and adequate Cre induction will be likely to allow us to have an RPS player <br />
<span class="name">E. coli</span> whose choice of either of rock, paper or scissors cannot be predicted.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/e/e6/Regulating_time_1.png" width="700px"/><br />
</div><br />
<br />
<p><br />
In our next experiments, we are going to vary the reaction time and the distance between the lox sites <br />
of each cassette. We believe precise modification of this two parameters must lead to our goal of <br />
making a randomizer in which each of the signaling molecules can be expressed with the same frequency <br />
(which results in each of the outcomes being expressed with the same probability).<br />
</p><br />
<br />
<h3 id="3.3">3.3 Survival of One strain</h3><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Image042.png" /><br />
</div><br />
<br />
<h4 id="3.3.1">3.3.1 Introduction: Minimal differences determine who will survive</h4><br />
<br />
<p><br />
In this section we will show a shocking scenario of evolution: <br />
the future of each of three different rival strains (whether the strain will die or survive) <br />
is marked by minimal differences between the initial population densities of the strains. <br />
Furthermore, we will also show that we can apply this very interesting result to create a randomizer <br />
that can be used in our Rock-Paper-Scissors game, due to the fact that only one of the rival strains <br />
will survive. More specifically, we assign to each of the three rival strains either of Rock, Paper or Scissors, <br />
make them compete for survival and take the surviving strain to represent the bacteria’s choice for the RPS game.<br />
</p><br />
<br />
<h4 id="3.3.2">3.3.2 Adjusting the Model to create a True Randomizer</h4><br />
<br />
<p><br />
The idea for creating this randomizer was born from a paper written in <br />
1996 by Durret and Levin. In it, the authors described a system of three types of bacteria that competed for <br />
survival in dynamic that resembled a Rock-Paper-Scissors (RPS) game. <br />
However, the model proposed in this paper is not fully appropriate for our RPS randomizer, <br />
since one of the three types of bacteria cannot ultimately survive <br />
(although it can dominate the system, i.e. have the highest population density, for definite periods of time). <br />
We will discuss more on the limitations we found in this model to be adopted as a <br />
randomizer and the modifications we made to create a true randomizer.<br />
</p><br />
<br />
<h4 id="3.3.3">3.3.3 How the Three Types of Bacteria Compete for Survival</h4><br />
<br />
<p><br />
The three types of bacteria that compete for survival use three tactics to outcompete their rivals: <br />
the production of a toxin (a bacteriocin called colicin) that is toxic to other strains, <br />
resistance to the toxin produced by other strains, and a higher birth rate than their rival strains. <br />
Namely, the three types of bacteria are: colicin-producing <span class="name">E. coli</span> (R), <br />
colicin-resistant <span class="name">E. coli</span> (P) <br />
and colicin-sensitive <span class="name">E. coli</span> (S). <br />
The colicin-producer outcompetes the colicin-sensitive by producing the colicin. The colicin-sensitive <br />
bacteria outcompetes the colicin-resistant because its birth rate is higher than that of the colicin-resistant. <br />
The colicin-resistant outcompetes the colicin producer because its birth rate is higher <br />
than that of the colicin producer. The colicin resistant bacteria are also able to produce colicin, <br />
but at a lower energetic cost, which allows them to have a higher birth rate.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bf/Image044.png" /><br />
</div><br />
<br />
<p><br />
The system was described by the following general differential equations<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/28/Colisin1.png" /><br />
</div><br />
<p><br />
Where<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c0/Colisin2.png" /><br />
</div><br />
<br />
<h4 id="3.3.4">3.3.4 The Old Model</h4><br />
<br />
<p><br />
In the model described by Durret and Levin’s paper the equations were as follows:<br />
</p><br />
<br />
<div align="center"><br />
<p>Producer</p><br />
<img src="https://static.igem.org/mediawiki/2011/1/1d/Colisin3.png" /><br /><br />
<p>Resistant</p><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Colisin4.png" /><br /><br />
<p>Sensitive</p><br />
<img src="https://static.igem.org/mediawiki/2011/3/36/Colisin5.png" /><br /><br />
</div><br />
<br />
<p><br />
These equations show that the colicin-resistant bacteria are completely immune to colicin <br />
(there is not death factor associated to colicin in the equation for du<sub>2</sub>/dt). <br />
However, as will be explained afterwards, this results in a loss of <br />
balance that does not allow building a true randomizing system.<br />
</p><br />
<p><br />
Now, setting the parameters as follows, the graph below was created by Durret and Levin.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/4/41/Colisin6.png" /><br /><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Colisin7.png" /><br />
<img src="http://partsregistry.org/wiki/images/b/bd/Modeling1.png" width="354px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/b/b1/Colisin8.png/800px-Colisin8.png" width="800px"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/5/55/Modeling2.png" width="354px"/><br /><br />
<br />
</div><br />
<br />
<h4 id="3.3.5">3.3.5 Our New Model</h4><br />
<p><br />
As mentioned before, the model proposed by Durret and Levin has critical limitations as a randomizer for the RPS game. <br />
To be able to create a true randomizer, we modified the differential equations of the model taking care to give it <br />
a biological meaning. With our new differential equations, any of the three types of bacteria can ultimately survive <br />
by outcompeting the other two strains, which will die. More specifically, we limited the resistance of the <br />
colicin-resistant bacteria in the sense that it would produce a type of bacteriocin that is only toxic to itself and <br />
to the sensitive strain, and additionally the resistant strain would also be vulnerable to the colicin produced by <br />
the colicin-producer. Since which strain will be the one that survives is determined by very small differences in <br />
the initial concentrations of the three different populations of bacteria, in practice this systems becomes a <br />
randomizer because of the imprecisions in the measurements that result, for example, when using micropipettes. <br />
This randomizer describes a new competition dynamic that could not be reproduced in the previous model proposed <br />
by Durret and Levin due to the instability along the<br />
<img src="https://static.igem.org/mediawiki/2011/e/e0/Image088.png" alt="u1">axis.<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c1/Colimodel1.png" alt="our new model" /><br />
</div><br />
<p><br />
If we set the parameters as follows<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/0/00/Colimodel2.png" alt="new model's coefficient" /><br />
</div><br />
<br />
<p><br />
and we graph this equations using a Matlab program, <br />
we get a graph which clearly shows there are stable points on each of the three axes <br />
(Figure 1, Up).<br />
</p><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b8/Model2.png" width="815px" style="float:center;"/><br /><br />
<img src="https://static.igem.org/mediawiki/2011/0/05/Image106.png" width="815px" style="float:center;"/><br />
</div><br />
<br />
<div align="center"><br />
Figure 1. Up: Our New model. Down: The Old Model<br />
</div><br />
<br />
<p><br />
These stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>) indicate that for the equations <br />
we have set all of the three strains may ultimately survive for infinite peiriods of time. <br />
The differences between our model and the model of Durret and Levin can be seen graphically <br />
in Figure 1. These graphs were plotted using Matlab. <br />
</p><br />
<p><br />
Note that the parameters we have set for our equations satisfy the initail conditions <br />
of the model proposed by Durret and Levin (indicated in black font) <br />
<img src="https://static.igem.org/mediawiki/2011/a/a4/Colimodel4.png" alt="new terms" width="800px"/><br />
</p><br />
<br />
<h4 id="3.3.6">3.3.6 The Biological Meaning of our Model</h4><br />
<br />
<p><br />
From a biological perspective, our model describes the existence of two strains of <br />
bacteria that produce two different types of bacteriocins. One of these strains is not <br />
completely resistant to its own bacteriocin nor to the bacteriocin produced by its <br />
rival strain. This can be justified as the consequence of insufficient/ineffective <br />
resistance protein production by the &ldquo;resistant&rdquo; strain. This limitation in the <br />
production of resistance protein could be thought of as a consequence of the <br />
&ldquo;resistant&rdquo; strain being a mutant of a colicin-sensitive strain.<br />
</p><br />
<br />
<h4 id="3.3.7">3.3.7 Making it Obvious</h4><br />
<br />
<p><br />
From the graph of our new model (Figure 1, left) it can be deduced that there are paths <br />
that converge at stable points (u<sub>1</sub>,0,0), (0,u<sub>2</sub>,0) and (0,0,u<sub>3</sub>), and that this paths all <br />
have an approximately common origin. In this section we would like to show that the <br />
origin of these paths is practically the same, and that in that sense we have designed <br />
a true randomizer (since, as mentioned before, the imprecisions that result in the <br />
experimental measurements will make it impossible to make the initial <br />
population density of the three strains the same). <br />
</p><br />
<br />
<p><br />
In the following set of graphs we will make it obvious that each of the three <br />
different strains of E. coli to survive in a random fashion by minimal <br />
differences on the initial concentrations of each strain.<br />
</p><br />
<p><br />
We modeled our results using Matlab. <br />
As can be seen in the graphs below, each of the strains can survive if their <br />
initial density in only tree hundredths (a.u.) greater than the other two strains' <br />
initial concentrations.<br />
</p><br />
<br />
<table><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/1/19/Image130.png/800px-Image130.png" alt="output1" Width="400px" /><br />
</a><br />
</td><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a1/Image132.png/800px-Image132.png" alt="output2" Width="400px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/1/11/Tt-modeling1.png" alt="coefficient of output1" Width="400px"/><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Tt-modeling2.png" alt="coefficient of output2" Width="400px"/><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png"><br />
<img src="https://static.igem.org/mediawiki/2011/thumb/a/a2/Image134.png/800px-Image134.png" alt="output3" Width="400px" /><br />
</a><br />
</td><br />
<td rowspan="2"><br />
With these graphs it becomes clear that the imprecisions in experimental measurements <br />
(i.e. pipetting) are enough to cause the outcome of Rock, Paper or Scissors signaling <br />
molecule to be random. Consequently, we can conclude that this randomizer is not <br />
only feasible but also practical and effective (let alone interesting).<br />
</td><br />
</tr><br />
<tr><br />
<td><img src="https://static.igem.org/mediawiki/2011/b/b7/Tt-modeling3.png" alt="coefficient of output3" Width="400px"/></td><br />
</tr><br />
</table><br />
<br />
<h2>References</h2><br />
<p><br />
<ul><br />
<li>Patrick C. Seed et al. "Activation of the Pseudomonas aeruginosa lasI Gene by LasR and the Pseudomonas Autoinducer PAI: an Autoinduction Regulatory Hierarchy" JOURNAL OF BACTERIOLOGY (1995)</li> <br />
<li>Shotaro Ayukawa et al. "Construction of a genetic AND gate under a new standard for assembly of genetic parts" BMC Genomics (2010)</li> <br />
<li>Robert Sidney Cox, III et al. "Programming gene expression with combinatorial promoters" molecular system biology (2007)</li> <br />
<li>Rolf Lutz et al. "Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements" Nucleic Acids Research (1997)</li> <br />
<li>Karina B.Xavier and Bonnie L.Bassler 2004. Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 238-248</li><br />
<li>Ting Xue, Liping Zhao, Haipeng Sun, Xianxuan Zhou, Baolon Sun 2009. LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing. Cell Research (2009), p.1258-1268</li><br />
<li>Liang Wang, Yoshifumi Hashimoto, Chen-Yu Tsao, James J.Valdes, and William E.Bentley 2004. Cyclic AMP(cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 2066-2076</li> <br />
<li>Jean Livet, Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, Robyn A. Bennis, Joshua R. Sanes and Jeff W. Lichtman (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature.</li><br />
<li>Kimi Araki, Masatake Araki1 and Ken-ichi Yamamura (2002). Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites, Nucleic Acids Research</li><br />
<li>Durret, R., Levin, S. Allelopathy in Spatially Distributed Populations (1997). Journal of Theoretical Biology, 185, 165-171. </li><br />
<br />
</ul><br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T06:28:11Z<p>Takuya 1613: </p>
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br /><br />
<p><br />
<big><b>How old are respondents?</b></big><br /><br />
<div align="left"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fb/Age-groups.png" width="350px" /><br /><br />
</div> <br />
</p><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T06:26:05Z<p>Takuya 1613: </p>
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<br />
<!-- page title --><br />
<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br /><br />
<p><br />
<big><b>How old are respondents?</b></big><br /><br />
<div align="left"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fb/Age-groups.png" width="400px" /><br /><br />
</div> <br />
</p><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T06:25:30Z<p>Takuya 1613: </p>
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<ul><br />
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<li><a href="#poster">3. Posters</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br /><br />
<p><br />
<big><b>How old are respondents?</b></big><br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fb/Age-groups.png" width="400px" /><br /><br />
</div> <br />
</p><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T06:25:02Z<p>Takuya 1613: </p>
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<h1> Human Practice </h1><br />
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<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
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<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
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is made and aimed by synthetic biology and iGEM.<br />
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used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
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<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br /><br />
<p><br />
<big><b>How old are respondents?</b></big><br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fb/Age-groups.png" width="600px" /><br /><br />
</div> <br />
</p><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/File:Age-groups.pngFile:Age-groups.png2011-10-28T06:22:21Z<p>Takuya 1613: </p>
<hr />
<div></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T06:04:09Z<p>Takuya 1613: </p>
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<li><a href="#poster">3. Posters</a></li><br />
<li><a href="#question">4. Questionnaire</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br />
</p><br /><br />
<p><br />
<big><b>How old are respondents?</b></big><br /> <br />
</p><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:56:54Z<p>Takuya 1613: </p>
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<!-- page title --><br />
<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
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<li id="menu_Home"><a href="https://2011.igem.org/Team:Tokyo_Tech">Home</a></li><br />
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<ul><br />
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htm">RPS-Game</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusions</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:43:58Z<p>Takuya 1613: </p>
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<li><a href="#question">4. Questionnaire</a></li><br />
<ul><br />
<li><a href="#4.1">4.1 All respondents</a></li><br />
<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusion</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:43:33Z<p>Takuya 1613: </p>
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<li><a href="#4.1">4.1 All respondents</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusion</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
<br />
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<ul><br />
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<li><a href="#poster">3. Posters</a></li><br />
<li><a href="#question">4. Questionnaire</a></li><br />
<ul><br />
<li><a href="#4.1">4.1 All respondents</a></li><br />
<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
<li><a href="#4.3">4.3 Respondents over 10 years old</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/57/All_of_human_Practice.png" alt="Result" width="800px" /><br /><br />
</div><br /><br /><br />
<br />
</p><br />
<h2 id="5.">5. Conclusion</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:41:15Z<p>Takuya 1613: </p>
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<li id="menu_Home"><a href="https://2011.igem.org/Team:Tokyo_Tech">Home</a></li><br />
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Project<br />
<ul><br />
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htm">RPS-Game</a></li><br />
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<h1> Human Practice </h1><br />
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<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
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<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
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<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
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<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
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<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/57/All_of_human_Practice.png" alt="Result" width="800px" /><br /><br />
</div><br /><br /><br />
<br />
</p><br />
<h2 id="5.">5. Conclusion</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:40:43Z<p>Takuya 1613: </p>
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<li><a href="#poster">3. Posters</a></li><br />
<li><a href="#question">4. Questionnaire</a></li><br />
<ul><br />
<li><a href="#4.1">4.1 All respondents</a></li><br />
<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/57/All_of_human_Practice.png" alt="Result" width="800px" /><br /><br />
</div><br /><br /><br />
<br />
</p><br />
<h2 id="5.">5. Conclusion</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:40:24Z<p>Takuya 1613: </p>
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<li><a href="#poster">3. Posters</a></li><br />
<li><a href="#question">4. Questionnaire</a></li><br />
<ul><br />
<li><a href="#4.1">4.1 All respondents</a></li><br />
<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
<li><a href="#4.3">4.3 Respondents over 10 years old</a></li><br />
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<!-- ############ Wrote main contents here ############### --><br />
<br />
<!-- page title --><br />
<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/57/All_of_human_Practice.png" alt="Result" width="600px" /><br /><br />
</div><br /><br /><br />
<br />
</p><br />
<h2 id="5.">5. Conclusion</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:38:28Z<p>Takuya 1613: </p>
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<li id="menu_Home"><a href="https://2011.igem.org/Team:Tokyo_Tech">Home</a></li><br />
<br />
<li id="menu_Project"><br />
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<ul><br />
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htm">RPS-Game</a></li><br />
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/index.htm">Make it Rain</a></li><br />
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<li><a href="#poster">3. Posters</a></li><br />
<li><a href="#question">4. Questionnaire</a></li><br />
<ul><br />
<li><a href="#4.1">4.1 All respondents</a></li><br />
<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
<li><a href="#4.3">4.3 Respondents over 10 years old</a></li><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br /><br />
</p><br />
<h2 id="5.">5. Conclusion</h2><br />
<p>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
<br />
</p><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:37:30Z<p>Takuya 1613: </p>
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<li><a href="#4.1">4.1 All respondents</a></li><br />
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<h1> Human Practice </h1><br />
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<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
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<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
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<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
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<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
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<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br /><br />
<h2 id="5.">5. Conclusion</h2><br />
Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!<br />
</p><br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:37:07Z<p>Takuya 1613: </p>
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<li><a href="https://2011.igem.org/Team:Tokyo_Tech/notebook">NoteBook</a></li><br />
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/team">Team</a></li><br />
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Sponsers.htm">Sponsors</a></li><br />
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Collaboration.htm">Collaboration</a></li><br />
</ul><br />
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<ul><br />
<li><a href="#Overview">1. OverView</a></li><br />
<li><a href="#card">2. The iGEM Card Game</a></li><br />
<li><a href="#poster">3. Posters</a></li><br />
<li><a href="#question">4. Questionnaire</a></li><br />
<ul><br />
<li><a href="#4.1">4.1 All respondents</a></li><br />
<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
<li><a href="#4.3">4.3 Respondents over 10 years old</a></li><br />
</ul><br />
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<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Respondents_over_10_years_old.png" alt="Result" width="600px" /><br /><br />
</div><br /><br /><br />
<h2 id="5.">5. Conclusion</h2><br />
<em>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!</em><br />
</p><br />
<br />
</p><br />
<br />
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</html></div>Takuya 1613http://2011.igem.org/Team:Tokyo_Tech/HumanPractice.htmTeam:Tokyo Tech/HumanPractice.htm2011-10-28T05:25:21Z<p>Takuya 1613: </p>
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<li><a href="#poster">3. Posters</a></li><br />
<li><a href="#question">4. Questionnaire</a></li><br />
<ul><br />
<li><a href="#4.1">4.1 All respondents</a></li><br />
<li><a href="#4.2">4.2 Respondents under 10 years old</a></li><br />
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<!-- page title --><br />
<h1> Human Practice </h1><br />
<br />
<p><br />
<h2 id="Overview">1. Overview</h2><br />
<p><br />
We love Synthetic Biology, and to share our passion with other people, we did educational activities as part of <br />
our human practices. We took part in an annual event called &ldquo;Manabi gate&rdquo;, which literally means &ldquo;learning <br />
gate&rdquo; in Japanese. Many universities and schools prepared activities for the participant.<br />
</p><br />
<p><br />
Even now, synthetic biology is not well known among people. But fragmental information can be spread, and it is <br />
typical reaction to feel &ldquo;the thing not knowing well is frightening&rdquo;. So we anyway would like to spread what <br />
is made and aimed by synthetic biology and iGEM.<br />
</p><br />
<p><br />
For this event, we created &ldquo;iGEM Card Game&rdquo;. One of the most important essences in synthetic biology is to <br />
combine genes. In our game, we reproduced the essence by combining cards. Before playing the game, posters were <br />
used for introduction. And after the game, we asked some questions for visitors.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Manabi-ppt2.png" alt="Creating Perception" /><br />
</div><br />
</p><br />
<br />
<br />
<h2 id="card">2. The iGEM Card Game</h2><br />
<p><br />
The iGEM card game has several series of cards. Each series consist of 3 to 5 cards each of which represents key parts of an iGEM project. The game’s rule is like poker. When we gather a whole series of cards, we can get points. By gathering them, the player can understand how the combination of the different genetic parts can be used to create living organisms with new and desired function. For example, one series represented iGEM 2009's Tokyo Tech project, namely the Terraforming of Mars project, which is referred by NASA Space Synthetic Biology.<br /><br />
<a href="https://2011.igem.org/NASA">https://2011.igem.org/NASA</a><br />
</p><br />
<p><br />
In particular, this series consists of three cards: &ldquo;anti-freezing protein&rdquo;, &ldquo;feeding from iron&rdquo;, and <br />
&ldquo;melanin production&rdquo;. The combination of the cards shows the project can make of Mars, a cold and inhospitable <br />
planet, a more habitable planet. Anti-freezing protein allows the bacteria to survive in Mars, as well as the <br />
iron-feeding ability. By producing melanin, bacteria become black and can help to rise the temperature of Mars.<br />
</p><br />
<p><br />
These cards are not only well representing nature of synthetic biology, but also have beautiful design. So the card game became very popular especially among children. Some children play the game many times and ask us “Can I bring these cards back?” Our card game attracts participants.<br />
</p><br />
<p><br />
To make sure they understood the essence of the game, we also further explained how the series which players <br />
had gathered represented an iGEM project.<br />
</p><br />
<br />
<br />
<h2 id="poster">3. Posters</h2><br />
<p><br />
We created three posters to introduce Synthetic Biology and iGEM to our audience. One of the posters was aimed <br />
at young audience, so we used Doraemon, a very famous comic character through many generations in Japan. The <br />
poster became very popular. In it, the characters make bacteria which can produce sweets, opposed to the <br />
depiction of bacteria as dirty living things as shown in detergent commercials. Other poster used metaphors and <br />
many illustrations to compare combining genetic parts with combining ingredients while cooking. A third poster <br />
was all about iGEM. This poster explained what iGEM is, and it served as a perfect way to invite our audience <br />
to play the &ldquo;iGEM Card Game&rdquo;. We think that our posters, as well as the card game, allowed our audience to <br />
know more about Synthetic Biology and iGEM.<br />
</p><br />
<br />
<h2 id="question">4. Questionnaire</h2><br />
<p><br />
<big><b>Our survey consisted of the following questions:</b></big><br /><br />
<br />
<ul style="list-style: none;"><br />
<li>Q1. Did you know microorganisms are used for making bread, soy sauce, etc.?</li><br />
<li>Q2. Did you know the words &ldquo;genetic engineering&rdquo;? </li><br />
<li>Q3. Did you know the words &ldquo;synthetic biology&rdquo;?</li><br />
<li>Q4. After hearing our explanations, what would you like to make using synthetic biology?</li><br />
<li>Q5. Did you become to be interested in synthetic biology? </li><br />
<li>Q6. Please feel free to write any other things, such as any comments or opinions you may have.</li><br />
</ul><br /><br />
<h3 id="4.1">4.1 All respondents</h3><br />
206 people answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/59/All_respondents.png" alt="Result" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.2">4.2 Respondents under 10 years old</h3><br />
97 people under 10 years old answered these questions. Here is the results we obtained.<br /><br />
<br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2011/5/56/Respondents_under_10_years_old.png" width="600px" /><br /><br />
</div><br /><br />
<h3 id="4.3">4.3 Respondents over 10 years old</h3><br />
95 people over 10 years old answered these questions. Here is the results we obtained.<br /><br />
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<em>Our results show that <big><b>many people of all ages</b></big> learned about and became interested in Synthetic Biology through our activity!</em><br />
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</html></div>Takuya 1613http://2011.igem.org/File:All_of_human_Practice.pngFile:All of human Practice.png2011-10-28T05:23:05Z<p>Takuya 1613: </p>
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<div></div>Takuya 1613http://2011.igem.org/File:Respondents_over_10_years_old.pngFile:Respondents over 10 years old.png2011-10-28T05:21:50Z<p>Takuya 1613: </p>
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<div></div>Takuya 1613http://2011.igem.org/File:Respondents_under_10_years_old.pngFile:Respondents under 10 years old.png2011-10-28T05:21:23Z<p>Takuya 1613: </p>
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<div></div>Takuya 1613