Team:Tokyo Tech

From 2010.igem.org

(Difference between revisions)
 
(102 intermediate revisions not shown)
Line 1: Line 1:
-
__NOTOC__
 
{{Tokyo_Tech_Template2}}
{{Tokyo_Tech_Template2}}
-
<html>
 
-
<body id=home onScroll="setMenu()">
 
<div id="super_main_wrapper">
<div id="super_main_wrapper">
-
<div id="tf_menu">
+
<div id="tf_menu">
-
<table border="1" cellspacing="4" cellpadding="0" id="fMENU" style="position:absolute;top:10px;left:10px;">
+
<font size="5" color="#eb8300"><b><center>Project menu</center></b></font>
-
<tr><th>[[[ MENU ]]]</th></tr>  
+
 
-
<tr><td><a href="http://www.impress.co.jp/">■Impress</a></td></tr>
+
<center>
-
<tr><td><a href="http://www.openspc2.org/" >■OpenSpace</a></td></tr>
+
<table id="table-01">
-
<tr><td><a href="http://www.yahoo.co.jp/" >■Yahoo</a></td></tr>
+
<tr>
-
<tr><td><a href="http://www.google.com/" >■Google</a></td></tr>  
+
<th>1 Graphic abstract  -YOU ARE HERE!- <br>
-
</table>  
+
</th>
-
</div> <!-- end tf_menu -->
+
</tr>
 +
<td>2 Apple reporter<br>
 +
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]
 +
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]
 +
</td>
 +
<tr>
 +
<td>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br>
 +
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]
 +
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]
 +
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]
 +
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br>
 +
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New series of P''ompC'']]
 +
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network]]
 +
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]
 +
</td>
 +
</tr>
 +
</table>
 +
</center>
 +
 
 +
<font size="3" color="#eb8300"><b>
 +
We won the track award, the Best Information Processing Project.<br>
 +
We also won the Gold Medal<br>
 +
Thank you all!!
 +
</b></font><br>
 +
[https://igem.org/Results?year=2010 See result page]
 +
 
 +
</div> <!-- tf_menu -->
<div id="tf_SubWrapper">
<div id="tf_SubWrapper">
-
Googleによるアクセス制限は、一般的には検索国の法律に従って行われるが、Googleはアメリカの企業であるため、アメリカ国内の法律によって違法と判断されたサイトについては、全世界で表示されない。例えば、デジタルミレニアム著作権法に抵触すると判断されたサイトについては、日本人向けのコンテンツであっても日本国内から検索出来ないし、アメリカ以外の国経由で検索しても同様である。
+
<font size="5"><b>1 Graphic abstract</b></font>
 +
__NOTOC__
 +
[[Image:Tokyotech_top5.png|center]]
-
中国国内のインターネットは政府によって検閲を受けており、中国版yahoo!、中国版Googleなど国際的サーチエンジンも例外ではない。「天安門事件」や「台湾」などの単語を検索しても政権に不都合なものは表示されなかった。また、「ダライ・ラマ14世」も禁止ワードに指定されており、チベット人の反感を買っていた。米国版Googleでは表示されるが、中国国内から中国版Google以外のGoogleにアクセスすることは出来なくなっているが、 Google側がこれ以上の検閲を行わない発表したため、政権に不都合なもの(天安門事件、ダライ・ラマ14世など)も表示されるようになった。それに加えて後述の中国国内からの攻撃も問題となり、Googleは中国から検索事業を撤退した。
 
-
2002年10月22日、およそ113のインターネット上のサイトがGoogleのドイツ語版とフランス語版から除去されているとの調査結果が報告された[40]。このサイト規制は主としてWhite Nationalistic、ナチ、反ユダヤ主義、イスラーム過激派のサイトに影響を与えた。フランスとドイツの法の下では、ヘイトスピーチとホロコーストの否定は違法である。Googleはこれらの法を遵守して、そのような題材を含むサイトを検索結果に含めないようにした。検索がこのような形で影響を受けているかどうか直接確認するすべは無い。
 
-
宗教団体サイエントロジーが同団体に批判的なサイトの削除をデジタル・ミレニアム著作権法 (DMCA) を根拠に求めたところGoogleは削除に応じた。その後Googleの姿勢を批判する市民グループが訴訟の動きを見せたことから元に戻した。
+
<font size="5" color="#ff8c00"><b>What do you think of humanity?</b></font><br>
-
個人名の検索結果の表示で名誉毀損に該当するか微妙なものについては、原則放置する方針をとっている。ただし、Google社員の個人名で検索した結果については、不都合な検索結果を表示しなかったり表示順位を下位に下げるなど、表示順位や検索結果表示について操作していると思われる例が散見される。
 
-
CNET出入り禁止事件
 
-
2005年7月、CNETのエリノア・ミルズ記者が執筆したGoogle絡みのプライバシー問題についての記事中、説明の一環として、CEOのエリック・シュミットについてGoogleで検索した結果を公表した。そこには、シュミットのおおよその資産や自宅住所、シュミットがGoogle株の売却を行ったことなどが掲載されていた[41]。 Google広報部は、この行為はプライバシー侵害に当たるとして、CNETの全サイトを検索結果から外した。さらに、CNETの記者全員からの取材を1 年間拒否するとの声明を出した。その2か月後に両者は和解しCNETのサイトは再び検索結果に表示されるようになった。
+
This is a common question that human beings have long sought to answer. And answer may vary.
-
グーグル八分
+
According to LONGMAN dictionary, humanity refers to,
 +
<blockquote>“the state of being human rather than an animal or machine. For example, kindness, respect and sympathy towards others.”</blockquote>
-
詳細は「グーグル八分」を参照
+
In the age of science and technology, people are getting more and more involved in their own business. Meanwhile, the society they are connected with is becoming limited to only a small group of people consisting their family and work partners. Moreover, their concerns are becoming more personal and less global.<br>
-
検索の際、検索エンジンスパムなどの検索妨害行為があるサイトや、各国の法律に照らし合わせてGoogleが違法と判断したサイトを、意図的に検索結果から除き、ユーザーが該当サイトのURLを検索できないようにすること、およびその対象となったサイトのこと。
+
In other words, humanity is losing its position in human life.
-
プライバシー問題
+
-
Googleではプライバシーを軽視する傾向があると言われており、Google Earth並びにGoogle マップで利用できる「公道のパノラマ写真が見ることができるストリートビュー」を公開して以降、Googleと一般人とのトラブルが絶えない。2008年8月5日から日本でも公開されたが、公開当日から個人のプライバシーを侵害しているとして日本国内より非難が集中し、のちに申告された物だけぼかしを入れたり画像をごっそり削除するなどの対処を行った[42]。ただし、いまだにプライバシーを侵害しているとして非難されている。また、「日本のGoogleでもプライバシーを軽視するような傾向である」ような発言を行ったGoogle社員もいる[43]。日本ではストリートビューを停止すべきとの要求も出された。これに対し、2009年6月日本の総務省は、適切な処理が行われている限りでは道路周辺映像提供サービスそのものに違法性はなく、一律の停止ではなく個別に侵害のおそれのある事案に対処していくことが望ましいとの見解をまとめた。一方で「法的な問題を克服できたからといって直ちに受け入れられるサービスといえるわけではない」と指摘しており、一般市民の抱く不安感の解消のための取組をサービス提供者に求めた[44][45][46]。
 
-
アメリカペンシルベニア州の住民がストリートビューで自宅内部を勝手に公開されたとして、Googleを相手に裁判を行っているが、その中でGoogleが答弁として「現代では完全なプライバシーなどは存在しない」と反論[47]、非難を浴びた。また、非営利組織のプライバシー保護団体がGoogle Earthを利用してGoogle取締役の自宅を公開した[48]。
+
<font size="4" color="#005396">Tokyo_Tech team has put its effort on producing E.coli with humanity,</font><br>hoping this to be awakening call for all of those who are forgetting their state of being human….
-
全ての検索結果の誤表示事故
+
-
2009年1月31日の23時30分頃(日本時間)から、検索サービス内でほぼ全ての検索結果に「このサイトはコンピュータに損害を与える可能性があります。」と表記され、リンク先にアクセスできなくなる不具合が発生した。翌2月1日0時25分(日本時間)頃には修正が行われ、復旧した[49]。ブログによるGoogle側の説明によれば、原因は「単純な人的ミス」によるもので、『有害サイトのフィルタリング更新中の手違いで、アドレス中にスラッシュ(/)の含まれる全てのリンクが「有害」とみなされた』という[50]。当初の発表では有害サイト情報を提供しているStopBadwareのミスとしていたが、StopBadwareの反論を受けて訂正する一幕もあった。
 
-
中国撤退騒動
 
-
2010年1月13日、中国で中国政府に批判的な政治活動家が所有するGMailアカウントに対して中国国内からInternet Explorerの脆弱性を利用した攻撃を受けていたことをGoogle公式ブログで告白、攻撃した一部ユーザーが中国政府であったため中国政府の検閲についても反発し中国から検索事業の撤退を示唆した[51][52]。これについて、中国外務省スポークスマンは「国内の法律に従うしかない」と述べるも、ヒラリー・クリントンアメリカ合衆国国務長官は「サイバー攻撃に対して説明を求める」とした[53]。なお、Internet Explorerはこの攻撃に使われた脆弱性が問題となり、オーストラリア政府機関が同攻撃に対する脆弱性が無い他ブラウザへの推奨を進めるといった異例の事態に発展、特にGoogleは中国ユーザーに利用者が多いInternet Explorer 6のブラウザに対してのサポートを同年3月で打ち切った[52]。
+
<h2>1. Concept -''E. coli'' with humanity-</h2><br>
 +
In order to make ''E. coli'' with humanity, we decided to engineer ''E. coli'' that can communicate with other, recognize the crisis situation other might be facing and show its sympathy toward it by rescuing it from dying...<br>
 +
Below we pictured our desired behavior of ''E. coli'' with humanity. Two kinds of cells are assumed. In normal conditions, they are competitors for survival because they are grown together in a single medium where the growing resources are limited. While in crisis conditions, such that one is dying, another cell would notice the crisis of dying cell and rescue it. The recovered cell appreciates the help by producing “apples”.
-
この事態を受けて中国政府と交渉を重ねたが[54]折り合いが付かず、2010年3月23日にGoogleは中国国内から検索事業を撤退、中国(google.cn)にアクセスすると検閲のない香港(google.com.hk)に飛ぶようになった。ただし、中国国内から香港の当該サイトで中国政府の規制しているキーワードを検索すると接続が出来なくなるなど、中国当局による規制が行われていると一部のメディアで報道された。[55][54]。
+
[[Image:Tokyotech_manga.jpg|center|640px|thumb|fig.1-1  1.They are competitors.  2.One is dying, another cell notices it.  3.Fine cell rescues dying cell. 4.Dying cell recovers and she gives "Apples" as expression of appreciation]]
-
</div> <!-- end tf_SubWrapper -->
+
-
<div class="clear"></div>
+
-
</div> <!-- end Super_main_wrapper -->
+
<h2>2. Apple reporters</h2><br>
 +
Our ''E. coli'' with humanity gives “apples” as expression of appreciation.
 +
It’s really difficult to make ''E. coli'' producing real apples. However, we succeeded in synthesizing two components of apples.
 +
<h3>2-1. Color -Carotenoid synthetic pathway complete!-</h3><br>
 +
[[Image:tokyotech_top1.JPG|250px|right]]This year, our team expanded the project of Cambridge 2009. We succeeded in synthesizing zeaxanthin by adding crtZ into crtEBIY likewise astaxanthin by crtZW. We identified them by TLC.
 +
Astaxanthin is the final metabolite of carotenoid synthetic pathway.
 +
[[Team:Tokyo_Tech/Project/Apple_Reporter|(See more…)]]
-
</body></html>
+
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<h3>2-2. Fragrance -We create Apple flavor in ''E. coli''-</h3><br>
 +
[[Image:tokyotech_top2.JPG|250px|right]]We designed apple fragrance expression device with MpAAT1. MpAAT1 is able to produce ester compounds with apple fragrance using some alcohols and Acetyl-CoA.
 +
[[Team:Tokyo_Tech/Project/Apple_Reporter2|(See more…)]]
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<h2>3. Artificial Cooperation System</h2><br>
 +
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing and chloramphenicol generator activated by LuxR are very important.
 +
First, we constructed and characterized several promoters regulated by LuxR and 3OC6HSL. We designed and constructed a new LuxR repression promoter. In addition, we characterized the functions of a LuxR repression promoter and a LuxR activation promoter already existed in the registry.
 +
Second, we constructed a new chloramphenicol resistance protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance gene generator survived in high concentration of chloramphenicol when 3OC6HSL exists.
 +
Third, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br>
 +
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|(See more…)]]
 +
<html>
 +
<center>
 +
<embed
 +
  src="https://static.igem.org/mediawiki/2010/5/53/Tokyotech_ACS.swf"
 +
  width="500"
 +
  height="375"
 +
/>
 +
</center>
 +
</html>
 +
 
 +
 
 +
<h3>3-1.  Lux repression promoter can work!</h3><br>
 +
In Artificial Cooperation System, two types of cells use quorum sensing to recognize population of the counterpart and help one another when they are dying. The quorum sensing in this system is regulated by AHL dependent transcriptional activation/repression. Therefore, we characterized activation/repression promoters. We examined the existing LuxR repression promoter which has never been characterized before in BioBrick registry. We found that the leaky expression of this promoter is too strong to use in this system. For this reason, we constructed and characterized a new LuxR repression promoter of which strength is weaker than the existing LuxR repression promoter.
 +
<br>
 +
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]
 +
[[Image:tokyotech_top3.JPG|250px|center]]
 +
 
 +
<h3>3-2.  Antibiotic Activation Device</h3><br>
 +
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (CmR) gene (BBa_K395162) which is activated by lux promoter. We found that the device is activated by LuxR/3OC6HSL complex and that the cell introduced the part was able to survive even in high chloramphenicol concentration (750 ug/ml) in the presence of 3OC6HSL. <br>
 +
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]
 +
[[Image:tokyotech_top4.JPG|250px|center]]
 +
 
 +
<h3>3-3.  ''luxI'' Assay</h3><br>
 +
In order to test whether rbs-LuxI-ter (K092400) works correctly, we measured the amount of 3OC6HSL synthesized by LuxI generator (K395146). We confirmed that rbs-LuxI-ter (K092400) worked as expected and the amount of 3OC6HSL was sufficient to induce the chloramphenicol resistance gene expression at maximum level, which is regulated by R0062 in the Artificial Cooperation System.
 +
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]
 +
[[Image:Tokyotech Fig.K395146-1.jpg|300px|center]]
 +
 
 +
<h3>3-4. mathematical modeling of  Artificial Cooperation System</h3><br>
 +
In order to confirm the feasibility of the Artificial Cooperation System, we simulated the system under typical four experimental conditions. First, when the system worked and any antibiotics did not exist, cell A and cell B in the system showed no difference of growth.<br>
 +
Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased.<br>
 +
Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline, which means it was rescued by Artificial Cooperation System.<br>
 +
Fourth, in the contrast, when the system worked and Kanamycin existed, the number of cell B increased after temporal decline. From the above results, the feasibility of the Artificial Cooperation System was confirmed.
 +
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]
 +
[[Image:Tokyotech cm sim result.jpg|250px|center]]
 +
 
 +
<h2>4. Full moon inhibits Artificial Cooperation system</h2><br>
 +
Have you heard the legend of 'The Wolfman'? <br>
 +
He is an ordinary man at daytime, but suddenly transforms into a ferocious wolf in the full-moon night. Our project aims to imitate the character of Wolfman. More specifically, we designed two types of ''E.coli'' that help each other to survive at daytime, whereas competing at full moon night. In order to create the “Wolfcoli”, we introduced " red-light-dependent gene expression network" and "band-detect network" and combined these networks with the Artificial Cooperation System. We characterized series of OmpC promoters and LacIM1 which are the crucial parts for our networks.
 +
[[Team:Tokyo_Tech/Project/wolf_coli|(See more…)]]
 +
[[Image:tokyotech_top6.JPG|250px|center]]
 +
 
 +
<h3>4-1. New series of osmoregulative promoters</h3><br>
 +
In order to fine tune the Wolfcoli system, we prepared a new series of ''OmpC'' promoter. The new series of promoters are P''ompC(C)'' [http://partsregistry.org/Part:BBa_K395301 BBa_K395301 ], P''ompC(CB)'' [http://partsregistry.org/Part:BBa_K395302 BBa_K395302 ]and P''ompC(CS1)'' [http://partsregistry.org/Part:BBa_K395303 BBa_K395303 ]. For measuring the strength of each promoter, we used GFP as a reporter. We have found that expression of GFP in ''OmpC(CB)'' and ''OmpC(CS1)'' promoters increased in high osmolarity medium. In contrast, under same conditions, there was no significant difference of GFP expression in ''OmpC(C)'' and ''OmpC(WT)'' promoters.
 +
[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|(See more…)]]
 +
[[Image:tokyotech_top7.JPG|250px|left]][[Image:tokyotech_top8.JPG|250px|left]]<br>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<h3>4-2. LacIMI showed weaker repression than WT</h3><br>
 +
We characterized LacIM1 (BBa_K082026), a mutation of LacIWT, which is a key component in the band-detect network. In fact, this part was registered by USTC(2008). However, sequence data of BBa_K082026 is incorrect and it was not well characterized in the BioBrick registry. Therefore, we registered this part as BBa_K395400 and confirmed that product of lacIM1 shows weaker repression to lac promoter than its wild type. In order to measure the function of LacI proteins, we constructed following two plasmids, BBa_K395401 (LacIM1) and BBa_K395402 (LacIWT), which have an arabinose inducible promoter. We measured GFP expression dependent on the input of arabinose and IPTG.[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|(See more…)]]
 +
[[Image:tokyotech_top9.JPG|250px|center]]
 +
 
 +
 
 +
 
 +
 
 +
<!--ここまで書いて良いですよ-->
 +
</div> <!-- end SubWrapper -->
 +
 
 +
 
 +
 
 +
</div> <!-- end Super_main_wrapper -->

Latest revision as of 22:34, 23 November 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

Project menu
1 Graphic abstract -YOU ARE HERE!-
2 Apple reporter
2-1 Color
2-2 Fragrance
3 Artificial Cooperation System
3-1 lux activation/repression promoter
3-2 resistance gene activation device
3-3 luxI Assay
3-4 modeling
4 Wolf coli overview
4-1 New series of PompC
4-2 lacIM1 for band-detect network
4-3 Wolf coli system

We won the track award, the Best Information Processing Project.
We also won the Gold Medal
Thank you all!!

See result page

1 Graphic abstract

Tokyotech top5.png


What do you think of humanity?


This is a common question that human beings have long sought to answer. And answer may vary. According to LONGMAN dictionary, humanity refers to,

“the state of being human rather than an animal or machine. For example, kindness, respect and sympathy towards others.”

In the age of science and technology, people are getting more and more involved in their own business. Meanwhile, the society they are connected with is becoming limited to only a small group of people consisting their family and work partners. Moreover, their concerns are becoming more personal and less global.

In other words, humanity is losing its position in human life.


Tokyo_Tech team has put its effort on producing E.coli with humanity,
hoping this to be awakening call for all of those who are forgetting their state of being human….


1. Concept -E. coli with humanity-


In order to make E. coli with humanity, we decided to engineer E. coli that can communicate with other, recognize the crisis situation other might be facing and show its sympathy toward it by rescuing it from dying...
Below we pictured our desired behavior of E. coli with humanity. Two kinds of cells are assumed. In normal conditions, they are competitors for survival because they are grown together in a single medium where the growing resources are limited. While in crisis conditions, such that one is dying, another cell would notice the crisis of dying cell and rescue it. The recovered cell appreciates the help by producing “apples”.

fig.1-1 1.They are competitors. 2.One is dying, another cell notices it. 3.Fine cell rescues dying cell. 4.Dying cell recovers and she gives "Apples" as expression of appreciation

2. Apple reporters


Our E. coli with humanity gives “apples” as expression of appreciation. It’s really difficult to make E. coli producing real apples. However, we succeeded in synthesizing two components of apples.

2-1. Color -Carotenoid synthetic pathway complete!-


Tokyotech top1.JPG
This year, our team expanded the project of Cambridge 2009. We succeeded in synthesizing zeaxanthin by adding crtZ into crtEBIY likewise astaxanthin by crtZW. We identified them by TLC.

Astaxanthin is the final metabolite of carotenoid synthetic pathway. (See more…)




2-2. Fragrance -We create Apple flavor in E. coli-


Tokyotech top2.JPG
We designed apple fragrance expression device with MpAAT1. MpAAT1 is able to produce ester compounds with apple fragrance using some alcohols and Acetyl-CoA.

(See more…)






3. Artificial Cooperation System


We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing and chloramphenicol generator activated by LuxR are very important. First, we constructed and characterized several promoters regulated by LuxR and 3OC6HSL. We designed and constructed a new LuxR repression promoter. In addition, we characterized the functions of a LuxR repression promoter and a LuxR activation promoter already existed in the registry. Second, we constructed a new chloramphenicol resistance protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance gene generator survived in high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.
(See more…)


3-1. Lux repression promoter can work!


In Artificial Cooperation System, two types of cells use quorum sensing to recognize population of the counterpart and help one another when they are dying. The quorum sensing in this system is regulated by AHL dependent transcriptional activation/repression. Therefore, we characterized activation/repression promoters. We examined the existing LuxR repression promoter which has never been characterized before in BioBrick registry. We found that the leaky expression of this promoter is too strong to use in this system. For this reason, we constructed and characterized a new LuxR repression promoter of which strength is weaker than the existing LuxR repression promoter.
(See more…)

Tokyotech top3.JPG

3-2. Antibiotic Activation Device


We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (CmR) gene (BBa_K395162) which is activated by lux promoter. We found that the device is activated by LuxR/3OC6HSL complex and that the cell introduced the part was able to survive even in high chloramphenicol concentration (750 ug/ml) in the presence of 3OC6HSL.
(See more…)

Tokyotech top4.JPG

3-3. luxI Assay


In order to test whether rbs-LuxI-ter (K092400) works correctly, we measured the amount of 3OC6HSL synthesized by LuxI generator (K395146). We confirmed that rbs-LuxI-ter (K092400) worked as expected and the amount of 3OC6HSL was sufficient to induce the chloramphenicol resistance gene expression at maximum level, which is regulated by R0062 in the Artificial Cooperation System. (See more…)

Tokyotech Fig.K395146-1.jpg

3-4. mathematical modeling of Artificial Cooperation System


In order to confirm the feasibility of the Artificial Cooperation System, we simulated the system under typical four experimental conditions. First, when the system worked and any antibiotics did not exist, cell A and cell B in the system showed no difference of growth.
Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased.
Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline, which means it was rescued by Artificial Cooperation System.
Fourth, in the contrast, when the system worked and Kanamycin existed, the number of cell B increased after temporal decline. From the above results, the feasibility of the Artificial Cooperation System was confirmed. (See more…)

Tokyotech cm sim result.jpg

4. Full moon inhibits Artificial Cooperation system


Have you heard the legend of 'The Wolfman'?
He is an ordinary man at daytime, but suddenly transforms into a ferocious wolf in the full-moon night. Our project aims to imitate the character of Wolfman. More specifically, we designed two types of E.coli that help each other to survive at daytime, whereas competing at full moon night. In order to create the “Wolfcoli”, we introduced " red-light-dependent gene expression network" and "band-detect network" and combined these networks with the Artificial Cooperation System. We characterized series of OmpC promoters and LacIM1 which are the crucial parts for our networks. (See more…)

Tokyotech top6.JPG

4-1. New series of osmoregulative promoters


In order to fine tune the Wolfcoli system, we prepared a new series of OmpC promoter. The new series of promoters are PompC(C) [http://partsregistry.org/Part:BBa_K395301 BBa_K395301 ], PompC(CB) [http://partsregistry.org/Part:BBa_K395302 BBa_K395302 ]and PompC(CS1) [http://partsregistry.org/Part:BBa_K395303 BBa_K395303 ]. For measuring the strength of each promoter, we used GFP as a reporter. We have found that expression of GFP in OmpC(CB) and OmpC(CS1) promoters increased in high osmolarity medium. In contrast, under same conditions, there was no significant difference of GFP expression in OmpC(C) and OmpC(WT) promoters. (See more…)

Tokyotech top7.JPG
Tokyotech top8.JPG








4-2. LacIMI showed weaker repression than WT


We characterized LacIM1 (BBa_K082026), a mutation of LacIWT, which is a key component in the band-detect network. In fact, this part was registered by USTC(2008). However, sequence data of BBa_K082026 is incorrect and it was not well characterized in the BioBrick registry. Therefore, we registered this part as BBa_K395400 and confirmed that product of lacIM1 shows weaker repression to lac promoter than its wild type. In order to measure the function of LacI proteins, we constructed following two plasmids, BBa_K395401 (LacIM1) and BBa_K395402 (LacIWT), which have an arabinose inducible promoter. We measured GFP expression dependent on the input of arabinose and IPTG.(See more…)

Tokyotech top9.JPG