Team:Tokyo Tech

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Latest revision as of 22:34, 23 November 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

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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

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!-

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-

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…)

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…)

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…)

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…)

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…)

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…)

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…)

@@ Line 8: / Line 8: @@
 <table id="table-01">
 <tr>
-<th>1 Graphical abstract  -YOUR ARE HERE!- <br>
+<th>1 Graphic abstract  -YOU ARE HERE!- <br>
 </th>
 </tr>
 <td>2 Apple reporter<br>
 :[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]
-::[[Team:Tokyo_Tech/Project/Apple_Reporter/Results|2-2-1 Apple color results]]
 :[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]
 </td>
@@ Line 25: / Line 24: @@
 </tr>
 <tr>
-<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br>
+<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br>
-:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]
+:[[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 wolfcoli system]]
+:[[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">
-<font size="5"><b>1 Graphical abstract</b></font>
+<font size="5"><b>1 Graphic abstract</b></font>
 __NOTOC__
-<h2>0. Project motivation ''-What is humanity?-''</h2><br>
+[[Image:Tokyotech_top5.png|center]]
-<font size="5" color="#ff8c00"><b>What do you think humanity is?</b></font><br>
-This is a common question which human have tried for long time. Answer might be different in each person.
-According to LONGMAN dictionary, humanity means, <br>
-“the state of being human rather than an animal or machine. For example, kindness, respect and sympathy towards others.”
-Then is it really impossible for the other livings to have those?<br>
+<font size="5" color="#ff8c00"><b>What do you think of humanity?</b></font><br>
-<font size="4" color="#1648e0">Tokyo Tech 2010 challenged this question.<br>We designed ''E. coli'' with humanity in order to prove that sympathetic doesn’t exist only in human!</font><br>
+This is a common question that human beings have long sought to answer. And answer may vary.
+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>
+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>
+In other words, humanity is losing its position in human life.
+<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….
 <h2>1. Concept -''E. coli'' with humanity-</h2><br>
-To make ''E. coli'' with humanity, we decided to engineer ''E. coli'' that behave kindly and have sympathy.<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 is a behavior of the ''E. coli'' with humanity we thought.
+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”.
-There are two kinds of cells. In normal conditions, they are competitors for survival. While in critical conditions, such that one is dying, another cell would notice the crisis of dying cell and help her.
-She gives him “apples” as a expression of her appeciation.
+[[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]]
-The relationship like this exists in human beings.
-[[Image:Tokyotech_top10.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 snd she gives "Apples" as expression of appreciation]]
 <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.
+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 could identify them by TLC.
+[[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…)]]
@@ Line 72: / Line 85: @@
 <h3>2-2. Fragrance -We create Apple flavor in ''E. coli''-</h3><br>
-[[Image:tokyotech_top2.JPG|250px|right]]''E. coli'' was modified to produces ester by addition of 2-MeBuOH or BuOH as a substrate.
+[[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.
-This ester gives off apple fragrance.
 [[Team:Tokyo_Tech/Project/Apple_Reporter2|(See more…)]]
@@ Line 85: / Line 97: @@
-<h2>3. Artificial Cooporation System</h2><br>
+<h2>3. Artificial Cooperation System</h2><br>
-<h3>3-1.  Genetic circiut of Artificial Cooperation System</h3><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.
-At section 1, we explained ''E. coli'' with humanity we want to create. We have already explained 4th illustration in the figure.
+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.
-Next move on to 1st to 3rd illustration. In order to realize the idea, we constructed a system that named “Artificial Cooperation System"
+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.
-There are two types of ''E. coli'' in this system. These two types of cells recognize each other by Quorum Sensing.
+Third, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br>
-CellA & CellB carry LasI gene and LuxI gene respectively. To see the rescuing process, click on the play button in the flash to start
 [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|(See more…)]]
 <html>
@@ Line 103: / Line 114: @@
-<h3>3-2.  Lux repression promoter can work!</h3><br>
+<h3>3-1.  Lux repression promoter can work!</h3><br>
-[[Image:tokyotech_top3.JPG|250px|right]]In order to construct Artificial Cooporation System, lux activation/repression device plays the key role.<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.
-Lux repression part has never been measured in iGEM while Lux activation part has measured by many iGEM teams.
+<br>
-This year we assayed the activity of lux repression promoter.
 [[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>
-<h3>3-3.  Antibiotic Activation Device</h3><br>
-[[Image:tokyotech_top4.JPG|250px|right]]We introduced chloramphenicol resistance  gene  the downstream of lux activation promoter. <br>
-This device activate in the presence of AHL. Signal molecule activates antibiotic resistance gene.
 [[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>
-[[Image:tokyotech_top5.JPG|250px|right]]We simulated Artificial Cooperation System and from this result, we predicted our system can work.
+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.
-<h2>4. Full moon inhibits Artificial Cooporation system</h2><br>
-[[Image:tokyotech_top6.JPG|250px|right]]In order to have a more intelligible& imaginable system, we tried to link our project to a famous& well-known character ”Wolfman”.<br>
-In our attempt to produce “wolfcoli”, we introduced light/switching function to the Artificial Cooperation System. Light sensitive promoter “PompC” and synthetic band detector circuit were used to perform function designed.
 [[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>
@@ Line 165: / Line 159: @@
-We introduced two new series of light sensitive promoters.
-[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|(See more…)]]
@@ Line 175: / Line 164: @@
 <h3>4-2. LacIMI showed weaker repression than WT</h3><br>
-[[Image:tokyotech_top9.JPG|250px|right]]We characterized the activity of lacIM1 which is necessary  for werewolf System. [[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|(See more…)]]
+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]]