Team:Tokyo Tech

From 2010.igem.org

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<blockquote>“the state of being human rather than an animal or machine. For example, kindness, respect and sympathy towards others.”</blockquote>
<blockquote>“the state of being human rather than an animal or machine. For example, kindness, respect and sympathy towards others.”</blockquote>
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In the age of science and technology, people are getting more and more involved in their own business, the society they are connected with, is becoming limited to only a small group of people consisting their family and work partners, their concerns are becoming more personal and less global, and in other words, humanity is losing its position in human life.  
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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.
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Moreover, their concerns are becoming more personal and less global.<br>
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<font size="4" color="#1648e0">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….</font><br>
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In other words, humanity is losing its position in human life.
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<font size="4" color="#1648e0">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….</font><br>
<h2>1. Concept -''E. coli'' with humanity-</h2><br>
<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>
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>
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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.<br>(They are called competitors; since they are grown together in a single medium and hence 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”.
+
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.(They are called competitors; since they are grown together in a single medium and hence 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”.
-
[[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 snd she gives "Apples" as expression of appreciation]]
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[[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 snd she gives "Apples" as expression of appreciation]]
<h2>2. Apple reporters</h2><br>
<h2>2. Apple reporters</h2><br>
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<h3>2-2. Fragrance -We create Apple flavor in ''E. coli''-</h3><br>
<h3>2-2. Fragrance -We create Apple flavor in ''E. coli''-</h3><br>
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[[Image:tokyotech_top2.JPG|250px|right]]''E. coli'' was modified to produces ester by addition of 2-MeBuOH or BuOH as a substrate.
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[[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.  
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These esters give off apple fragrance.
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[[Team:Tokyo_Tech/Project/Apple_Reporter2|(See more…)]]
[[Team:Tokyo_Tech/Project/Apple_Reporter2|(See more…)]]
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<h2>3. Artificial Cooporation System</h2><br>
<h2>3. Artificial Cooporation System</h2><br>
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At section 1, we explained ''E. coli'' with humanity we want to create. We have already explained 4th illustration in the figure.  
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We built Artificial Cooperation System to engineered E.coli with humanity. In this system quorum sensing and chloramphenicol generator activated by LuxR are very important. <br>
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Next move on to 1st to 3rd illustration. In order to realize the idea, we constructed a system that named “Artificial Cooperation System"
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First, we constructed and characterized several promoters regulated by LuxR and 3OC6HSL. We designed and constructed a new LuxR repression promoter. Also, we characterized the functions of a LuxR repression promoter and a LuxR activation promoter in the registry.<br>
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There are two types of ''E. coli'' in this system. These two types of cells recognize each other by Quorum Sensing.  
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Second, we constructed a new chloramphenicol resistance gene generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance gene generator under high concentration of chloramphenicol when 3OC6HSL exists.<br>
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CellA & CellB carry LasI gene and LuxI gene respectively. To see the rescuing process, click on the play button in the flash to start
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Third, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br>
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|(See more…)]]
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|(See more…)]]
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<h3>3-1.  Lux repression promoter can work!</h3><br>
<h3>3-1.  Lux repression promoter can work!</h3><br>
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[[Image:tokyotech_top3.JPG|250px|right]]In order to construct Artificial Cooporation System, lux activation/repression device plays the key role.<br>
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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. Even though the GFP expression was repressed in the presence of AHL, cell growth is inhibited because of the overexpression of GFP occurred in the absence of AHL. For this reason, we designed and constructed a new repression promoter that regulates the transcription appropriately dependent on the signal input.
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Lux repression part has never been measured in iGEM while Lux activation part has measured by many iGEM teams.  
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<br>
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This year we assayed the activity of lux repression promoter.
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[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]
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[[Image:tokyotech_top3.JPG|250px|center]]
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<h3>3-2.  Antibiotic Activation Device</h3><br>
<h3>3-2.  Antibiotic Activation Device</h3><br>
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[[Image:tokyotech_top4.JPG|250px|right]]We introduced chloramphenicol resistance gene the downstream of lux activation promoter. <br>
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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>
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We found that the chloramphenicol resistance gene was activated by LuxR/3OC6HSL complex and that the cell introduced the device was able to survive even in high chloramphenicol concentration (750 ug/ml) in the presence of 3OC6HSL.  
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[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]
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[[Image:tokyotech_top4.JPG|250px|center]]
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<h3>3-3.  ''lux''I Assay</h3><br>
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In order to test whether rbs-LuxI-term (K092400) works correctly, we measured the amount of 3OC6HSL synthesized by LuxI generator (K395146). We confirmed that rbs-LuxI-term (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.
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[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]
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[[Image:tokyotech_top5.JPG|250px|center]]
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<h3>3-4. mathematical modeling of  Artificial Cooperation System</h3><br>
<h3>3-4. mathematical modeling of  Artificial Cooperation System</h3><br>
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[[Image:tokyotech_top5.JPG|250px|right]]We simulated Artificial Cooperation System and from this result, we predicted our system can work.
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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>
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Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased.<br>
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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>
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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…)]]
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]
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[[Image:tokyotech_top5.JPG|250px|center]]
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<h2>4. Full moon inhibits Artificial Cooporation system</h2><br>
<h2>4. Full moon inhibits Artificial Cooporation system</h2><br>
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[[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>
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Have you heard the legend of 'The Wolfman'? <br>
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In our attempt to produce “wolfcoli”, we introduced light/switching function to the Artificial Cooperation System. Light sensitive promoter “P''omp''C” and synthetic band-detect network were used to perform function designed.
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They're ordinary man at daytime, but suddenly transform into a ferocious wolf in the full-moon night. Our project aim to imitate the character of Wolfman, more specifically, designing two types of ''E.coli'' that helps each other to survive at daytime, whereas competing at full moon night. In order to create the “Wolf coli”, we introduced " red-light-dependent gene expression network" and "band-detect network" and combined these networks with the Artificial Cooperation System. We conducted experiment on series of OmpC promoter, and LacIM1 which are crucial parts of our networks.  
[[Team:Tokyo_Tech/Project/wolf_coli|(See more…)]]
[[Team:Tokyo_Tech/Project/wolf_coli|(See more…)]]
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[[Image:tokyotech_top6.JPG|250px|center]]
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<h3>4-1. New series of osmoregulative promoters</h3><br>
<h3>4-1. New series of osmoregulative promoters</h3><br>
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We introduced two new series of osmoregulative promoters into red-light-dependent gene expression network.[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|(See more…)]]  
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In order to fine tune the Wolfcoli system. We prepared a series of ''Omp''C promoter. The new series of promoters are P''omp''C(C) BBa_K395301 , P''omp''C(CB) BBa_K395302 and P''omp''C(CS1) BBa_K395303 . In order to measure the strength of each promoter, we used GFP as a reporter. We have found that expression of GFP in ''Omp''C(CB) and ''Omp''C(CS1) promoters increased in high osmolarity medium. In contrast, under same conditions, there was no significant difference of GFP expression in ''Omp''C(C) and ''Omp''C(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>
[[Image:tokyotech_top7.JPG|250px|left]][[Image:tokyotech_top8.JPG|250px|left]]<br>
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<h3>4-2. LacIMI showed weaker repression than WT</h3><br>
<h3>4-2. LacIMI showed weaker repression than WT</h3><br>
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[[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…)]]
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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 Plac 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…)]]
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[[Image:tokyotech_top9.JPG|250px|center]]

Revision as of 20:49, 27 October 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

1 Graphical abstract

Tokyotech whatishumanity.jpg

What do you think humanity is?


This is a common question that human have been facing for a long time. Answers might differ from person to person. Looking up the word “humanity” in LONGMAN dictionary, it was described as

“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.(They are called competitors; since they are grown together in a single medium and hence 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 snd 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 could identify 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 Cooporation System


We built Artificial Cooperation System to engineered 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. Also, we characterized the functions of a LuxR repression promoter and a LuxR activation promoter in the registry.
Second, we constructed a new chloramphenicol resistance gene generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance gene generator under 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. Even though the GFP expression was repressed in the presence of AHL, cell growth is inhibited because of the overexpression of GFP occurred in the absence of AHL. For this reason, we designed and constructed a new repression promoter that regulates the transcription appropriately dependent on the signal input.
(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-term (K092400) works correctly, we measured the amount of 3OC6HSL synthesized by LuxI generator (K395146). We confirmed that rbs-LuxI-term (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 top5.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 sell 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 top5.JPG

4. Full moon inhibits Artificial Cooporation system


Have you heard the legend of 'The Wolfman'?
They're ordinary man at daytime, but suddenly transform into a ferocious wolf in the full-moon night. Our project aim to imitate the character of Wolfman, more specifically, designing two types of E.coli that helps each other to survive at daytime, whereas competing at full moon night. In order to create the “Wolf coli”, we introduced " red-light-dependent gene expression network" and "band-detect network" and combined these networks with the Artificial Cooperation System. We conducted experiment on series of OmpC promoter, and LacIM1 which are crucial parts of 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 series of OmpC promoter. The new series of promoters are PompC(C) BBa_K395301 , PompC(CB) BBa_K395302 and PompC(CS1) BBa_K395303 . In order to measure 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 Plac 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