http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Kitano&year=&month=2010.igem.org - User contributions [en]2024-03-28T13:54:15ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-28T03:59:29Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to endow E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to activate chloramphenicol resistance gene. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. In our idea of ''E. coli'' with humanity, there are two types 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. For realization of our idea, we constructed a system that named “Artificial Cooperation System”.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. In cell A (Fig 3-0-2), production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B (Fig3-0-3), production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. On the other hand, 3OC12HSL activates resistance gene of cell B and 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
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<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
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<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062, both of which are existing parts. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), GFP expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, GFP expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, ''E. coli'' with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_TechTeam:Tokyo Tech2010-10-28T03:58:30Z<p>Kitano: </p>
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<th>1 Graphic abstract -YOU ARE HERE!- <br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<td>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New series of P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>1 Graphic abstract</b></font><br />
__NOTOC__<br />
[[Image:Tokyotech_top5.png|center]]<br />
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<font size="5" color="#ff8c00"><b>What do you think of humanity?</b></font><br><br />
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This is a common question that human beings have long sought to answer. And answer may vary. <br />
According to LONGMAN dictionary, humanity refers to,<br />
<blockquote>“the state of being human rather than an animal or machine. For example, kindness, respect and sympathy towards others.”</blockquote><br />
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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. Moreover, their concerns are becoming more personal and less global.<br><br />
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In other words, humanity is losing its position in human life.<br />
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<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….<br />
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<h2>1. Concept -''E. coli'' with humanity-</h2><br><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><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”.<br />
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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 and she gives "Apples" as expression of appreciation]]<br />
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<h2>2. Apple reporters</h2><br><br />
Our ''E. coli'' with humanity gives “apples” as expression of appreciation.<br />
It’s really difficult to make ''E. coli'' producing real apples. However, we succeeded in synthesizing two components of apples. <br />
<h3>2-1. Color -Carotenoid synthetic pathway complete!-</h3><br><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.<br />
Astaxanthin is the final metabolite of carotenoid synthetic pathway.<br />
[[Team:Tokyo_Tech/Project/Apple_Reporter|(See more…)]]<br />
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<h3>2-2. Fragrance -We create Apple flavor in ''E. coli''-</h3><br><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. <br />
[[Team:Tokyo_Tech/Project/Apple_Reporter2|(See more…)]]<br />
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<h2>3. Artificial Cooperation System</h2><br><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.<br />
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.<br />
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.<br />
Third, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br><br />
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|(See more…)]]<br />
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<h3>3-1. Lux repression promoter can work!</h3><br><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 />
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[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
[[Image:tokyotech_top3.JPG|250px|center]]<br />
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<h3>3-2. Antibiotic Activation Device</h3><br><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><br />
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
[[Image:tokyotech_top4.JPG|250px|center]]<br />
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<h3>3-3. ''luxI'' Assay</h3><br><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.<br />
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
[[Image:Tokyotech Fig.K395146-1.jpg|300px|center]]<br />
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<h3>3-4. mathematical modeling of Artificial Cooperation System</h3><br><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><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><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><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. <br />
[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[Image:Tokyotech cm sim result.jpg|250px|center]]<br />
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<h2>4. Full moon inhibits Artificial Cooperation system</h2><br><br />
Have you heard the legend of 'The Wolfman'? <br><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. <br />
[[Team:Tokyo_Tech/Project/wolf_coli|(See more…)]]<br />
[[Image:tokyotech_top6.JPG|250px|center]]<br />
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<h3>4-1. New series of osmoregulative promoters</h3><br><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.<br />
[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|(See more…)]] <br />
[[Image:tokyotech_top7.JPG|250px|left]][[Image:tokyotech_top8.JPG|250px|left]]<br><br />
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<h3>4-2. LacIMI showed weaker repression than WT</h3><br><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…)]]<br />
[[Image:tokyotech_top9.JPG|250px|center]]<br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_repTeam:Tokyo Tech/Project/Artificial Cooperation System/lux act rep2010-10-28T03:23:28Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:3-1 ''lux'' activation/repression promoter -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New series of P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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=''lux'' activation/repression promoter=<br />
=Abstract=<br />
In Artificial Cooperation System, two types of cells use quorum sensing to recognize population of the counterpart and to help the counterparts 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 rate decreased 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. <br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 LuxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3 LuxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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=Introduction=<br />
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In Synthetic Biology, transcription activation is used frequently. Transcription repression by using AHL is also important, however, the device which has delay for transcription/translation through inverter is used a lot in this case. We decided to experience LuxR repression promoter for the quick response of signal dependent repression.<br><br />
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=Result=<br />
==R0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)==<br />
First, we characterized R0062, the well-known LuxR activation promoter in order to establish our Tokyo_Tech team experimental system for Artificial Cooperation System.<br><br />
The expression of GFP with 100nM 3OC6HSL around 30-folds increased comparing with the expression without 3OC6HSL.<br><br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 LuxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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We confirmed fluorescence intensity of LuxR activation promoter is dependent on 3OC6HSL concentration. The AHL concentration which shows half of maximam activity is less than 5nM. <br><br />
[[IMAGE:tokyotech_LuxR ractivatio promoter assay2.jpg|400px|left|thumb|fig.3-1-2 Fluorescence intensity dependent on the concentration of AHL (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==R1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)==<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3 LuxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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===R1-1, R0061 (promoter repressed by LuxR/3OC6HSL)===<br />
Next, we characterized the existing part R0061, LuxR repression promoter. We examined whether the amount of transcription is appropriate when signal is off and how much this promoter represses. <br><br />
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL. <br />
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===R1-2, K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak===<br />
We confirmed R0061 and found increase of cells was inhibited due to a high level of expression although it is repressed by AHL. Therefore, we designed a new appropriate promoter by changing one base of R0061.<br><br />
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL. We found the level of expression is appropriate and this promoter work as expected.<br />
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=Conclusion=<br />
We designed the new promoter which is repressed LuxR/3OC6HSL complex by changing one base of existing promoter. We confirmed this promoter works as we expected.<br><br />
It is not so difficult to make the promoter which strength is between these two by designing.<br />
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=Material & Methods=<br />
==M0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)==<br />
===fluorescence intensity in the presence/absence of AHL ===<br />
We constructed K395100 combining R0062 and K121013. K121013 is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) on pSB6A1. S03119 is a LuxR generator which is regulated by PTetR, which is repressed by TetR. In this experiment, we don’t use TetR, so S03119 functions as a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.<br />
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control. <br />
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[[IMAGE:Tokyotech_R0062assay_construction.png|400px]]<br />
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*samples<br />
#[Plux act - ''gfp''](BBa_K395100) on pSB6A1 + [PtetR – LuxR] on pSB3K3<br />
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
#negative control:. [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
*Strain<br />
DH5&alpha;<br />
*protocol<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.60.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.<br />
#Induction for 3 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
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===fluorescence intensity dependent on [AHL]===<br />
*samples<br />
#[Plux act - ''gfp''](BBa_K395100) on pSB6A1 + [ptet – LuxR] on pSB3K3<br />
*Strain<br />
DH5&alpha;<br />
*protocol<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture) Prepare the same 7 tubes for each sample.<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.60.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture. The final concentration of 3OC6HSL is 1, 3, 5, 10, 30, 50, 100nM.<br />
#Induction for 3 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
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==M1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)==<br />
===M1-1, characterization of R0061 (promoter repressed by LuxR/3OC6HSL)===<br />
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We constructed K395101 combining R0061 and K121013, which is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) on pSB6A1. S03119 is a LuxR generator which is repressed by TetR. In this experiment, we don’t use TetR, therefore, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.<br />
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control.<br />
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[[IMAGE:Tokyotech_R0061assay_construction.png|400px]]<br />
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*'''samples'''<br />
#[Plux rep - ''gfp''](BBa_K395101) on pSB6A1 + [PtetR – LuxR] on pSB3K3<br />
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
#negative control: [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3<br />
*'''Strain'''<br />
DH5&alpha;<br />
*'''protocol'''<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan).(→fresh culture)<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.80.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of AHL is 100nM.<br />
#Induction for 2 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
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===M1-2, characterization of K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak===<br />
We constructed K395105 combining K395008 and K121013. K121013 is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) and this backbone is pSB6A1. Promoter of S03119 is PtetR, which is repressed by tetR. In this experiment, we don’t use TetR, so, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.<br />
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control.<br />
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[[IMAGE:Tokyotech_K395008assay_construction.png|400px]]<br />
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*samples<br />
#[R0061weak - ''gfp''](BBa_K395105) on pSB6A1 + [PtetR – LuxR] on pSB3K3<br />
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR]) on pSB3K3 <br />
#negative control: [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
*Strain<br />
DH5&alpha;<br />
*protocol<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.60.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.<br />
#Induction for 3 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
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=Reference=<br />
#KRISTI A. EGLAND & E. P. GREENBERG, Conversion of the Vibrio fischeri Transcriptional Activator LuxR, to a Repressor. JOURNAL OF BACTERIOLOGY, Feb. 2000, p. 805–811<br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_repTeam:Tokyo Tech/Project/Artificial Cooperation System/lux act rep2010-10-28T03:21:15Z<p>Kitano: </p>
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<tr><br />
<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:3-1 ''lux'' activation/repression promoter -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
</th><br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New series of P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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=''lux'' activation/repression promoter=<br />
=Abstract=<br />
In Artificial Cooperation System, two types of cells use quorum sensing to recognize population of the counterpart and to help the counterparts 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 rate decreased 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. <br />
<br><br />
<br />
<br />
[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 LuxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3 LuxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
<br />
=Introduction=<br />
<br />
In Synthetic Biology, transcription activation is used frequently. Transcription repression by using AHL is also important, however, the device which has delay for transcription/translation through inverter is used a lot in this case. We decided to experience LuxR repression promoter for the quick response of signal dependent repression.<br><br />
<br />
=Result=<br />
==R0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)==<br />
First, we characterized R0062, the well-known LuxR activation promoter in order to establish our Tokyo_Tech team experimental system for Artificial Cooperation System.<br><br />
The expression of GFP with 100nM 3OC6HSL around 30-folds increased comparing with the expression without 3OC6HSL.<br><br />
<br />
[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 LuxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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We confirmed fluorescence intensity of LuxR activation promoter is dependent on 3OC6HSL concentration. The AHL concentration which shows half of maximam activity is less than 5nM. <br><br />
[[IMAGE:tokyotech_LuxR ractivatio promoter assay2.jpg|400px|left|thumb|fig.3-1-2 Fluorescence intensity dependent on the concentration of AHL (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==R1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)==<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3 LuxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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===R1-1, R0061 (promoter repressed by LuxR/3OC6HSL)===<br />
Next, we characterized the existing part R0061, LuxR repression promoter. We examined whether the amount of transcription is appropriate when signal is off and how much this promoter represses. <br><br />
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL. <br />
<br />
===R1-2, K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak===<br />
We confirmed R0061 and found increase of cells was inhibited due to a high level of expression although it is repressed by AHL. Therefore, we designed a new appropriate promoter by changing one base of R0061.<br><br />
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL. We found the level of expression is appropriate and this promoter work as expected.<br />
<br />
=Conclusion=<br />
We designed the new promoter which is repressed LuxR/3OC6HSL complex by changing one base of existing promoter. We confirmed this promoter works as we expected.<br><br />
It is not so difficult to make the promoter which strength is between these two by designing.<br />
<br />
=Material & Methods=<br />
==M0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)==<br />
===fluorescence intensity in the presence/absence of AHL ===<br />
We constructed K395100 combining R0062 and K121013. K121013 is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) on pSB6A1. S03119 is a LuxR generator which is regulated by PTetR, which is repressed by TetR. In this experiment, we don’t use TetR, so S03119 functions as a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.<br />
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control. <br />
<br />
[[IMAGE:Tokyotech_R0062assay_construction.png|400px]]<br />
<br />
*samples<br />
#[Plux act - ''gfp''](BBa_K395100) on pSB6A1 + [PtetR – LuxR] on pSB3K3<br />
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
#negative control:. [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
*Strain<br />
DH5&alpha;<br />
*protocol<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.60.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.<br />
#Induction for 3 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
<br />
===fluorescence intensity dependent on [AHL]===<br />
*samples<br />
#[Plux act - ''gfp''](BBa_K395100) on pSB6A1 + [ptet – LuxR] on pSB3K3<br />
*Strain<br />
DH5&alpha;<br />
*protocol<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture) Prepare the same 7 tubes for each sample.<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.60.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture. The final concentration of 3OC6HSL is 1, 3, 5, 10, 30, 50, 100nM.<br />
#Induction for 3 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
<br />
==M1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)==<br />
===M1-1, characterization of R0061 (promoter repressed by LuxR/3OC6HSL)===<br />
<br />
We constructed K395101 combining R0061 and K121013, which is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) on pSB6A1. S03119 is a LuxR generator which is repressed by TetR. In this experiment, we don’t use TetR, therefore, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.<br />
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control.<br />
<br />
[[IMAGE:Tokyotech_R0061assay_construction.png|400px]]<br />
<br />
*'''samples'''<br />
#[Plux rep - ''gfp''](BBa_K395101) on pSB6A1 + [PtetR – LuxR] on pSB3K3<br />
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
#negative control: [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3<br />
*'''Strain'''<br />
DH5&alpha;<br />
*'''protocol'''<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan).(→fresh culture)<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.80.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of AHL is 100nM.<br />
#Induction for 2 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
<br />
===M1-2, characterization of K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak===<br />
We constructed K395105 combining K395008 and K121013. K121013 is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) and this backbone is pSB6A1. Promoter of S03119 is PtetR, which is repressed by tetR. In this experiment, we don’t use TetR, so, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.<br />
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control.<br />
<br />
[[IMAGE:Tokyotech_K395008assay_construction.png|400px]]<br />
<br />
*samples<br />
#[R0061weak - ''gfp''](BBa_K395105) on pSB6A1 + [PtetR – LuxR] on pSB3K3<br />
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR]) on pSB3K3 <br />
#negative control: [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
*Strain<br />
DH5&alpha;<br />
*protocol<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.60.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.<br />
#Induction for 3 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
<br />
=Reference=<br />
#KRISTI A. EGLAND & E. P. GREENBERG, Conversion of the Vibrio fischeri Transcriptional Activator LuxR, to a Repressor. JOURNAL OF BACTERIOLOGY, Feb. 2000, p. 805–811<br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_repTeam:Tokyo Tech/Project/Artificial Cooperation System/lux act rep2010-10-28T03:19:51Z<p>Kitano: /* R0, characterization of R0062 (promoter activated by LuxR/3OC6HSL) */</p>
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:3-1 ''lux'' activation/repression promoter -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New series of P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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=''lux'' activation/repression promoter=<br />
=Abstract=<br />
In Artificial Cooperation System, two types of cells use quorum sensing to recognize population of the counterpart and to help the counterparts 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 rate decreased 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. <br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 LuxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3 LuxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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=Introduction=<br />
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In Synthetic Biology, transcription activation is used frequently. Transcription repression by using AHL is also important, however, the device which has delay for transcription/translation through inverter is used a lot in this case. We decided to experience LuxR repression promoter for the quick response of signal dependent repression.<br><br />
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=Result=<br />
==R0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)==<br />
First, we characterized R0062, the well-known LuxR activation promoter in order to establish our Tokyo_Tech team experimental system for Artificial Cooperation System.<br><br />
The expression of GFP with 100nM 3OC6HSL around 30-folds increased comparing with the expression without 3OC6HSL.<br><br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 LuxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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We confirmed fluorescence intensity of LuxR activation promoter is dependent on 3OC6HSL concentration. The AHL concentration which shows half of maximam activity is less than 5nM. <br><br />
[[IMAGE:tokyotech_LuxR ractivatio promoter assay2.jpg|400px|left|thumb|fig.3-1-2 Fluorescence intensity dependent on the concentration of AHL (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==R1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)==<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3 LuxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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===R1-1, R0061 (promoter repressed by LuxR/3OC6HSL)===<br />
Next, we characterized the existing part R0061, LuxR repression promoter. We examined whether the amount of transcription is appropriate when signal is off and how much this promoter represses. <br><br />
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL. <br />
<br />
===R1-2, K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak===<br />
We confirmed R0061 and found increase of cells was inhibited due to a high level of expression although it is repressed by AHL. Therefore, we designed a new appropriate promoter by changing one base of R0061.<br><br />
The expression of GFP with 100nM 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL. We found the level of expression is appropriate and this promoter work as expected.<br />
<br />
=Conclusion=<br />
We designed the new promoter which is repressed LuxR/3OC6HSL complex by changing one base of existing promoter. We confirmed this promoter works as we expected.<br><br />
It is not so difficult to make the promoter which strength is between these two by designing.<br />
<br />
=Material & Methods=<br />
==M0, characterization of R0062 (promoter activated by LuxR/3OC6HSL)==<br />
===fluorescence intensity in the presence/absence of AHL ===<br />
We constructed K395100 combining R0062 and K121013. K121013 is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) on pSB6A1. S03119 is a LuxR generator which is regulated by PTetR, which is repressed by TetR. In this experiment, we don’t use TetR, so S03119 functions as a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.<br />
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control. <br />
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[[IMAGE:Tokyotech_R0062assay_construction.png|400px]]<br />
<br />
*samples<br />
#[Plux act - ''gfp''](BBa_K395100) on pSB6A1 + [PtetR – LuxR] on pSB3K3<br />
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
#negative control:. [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
*Strain<br />
DH5&alpha;<br />
*protocol<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.60.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.<br />
#Induction for 3 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
<br />
===fluorescence intensity dependent on [AHL]===<br />
*samples<br />
#[Plux act - ''gfp''](BBa_K395100) on pSB6A1 + [ptet – LuxR] on pSB3K3<br />
*Strain<br />
DH5&alpha;<br />
*protocol<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture) Prepare the same 7 tubes for each sample.<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.60.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture. The final concentration of 3OC6HSL is 1, 3, 5, 10, 30, 50, 100nM.<br />
#Induction for 3 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
<br />
==M1, characterization of R0061 & K395008 (promoter repressed by LuxR/3OC6HSL)==<br />
===M1-1, characterization of R0061 (promoter repressed by LuxR/3OC6HSL)===<br />
<br />
We constructed K395101 combining R0061 and K121013, which is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) on pSB6A1. S03119 is a LuxR generator which is repressed by TetR. In this experiment, we don’t use TetR, therefore, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.<br />
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control.<br />
<br />
[[IMAGE:Tokyotech_R0061assay_construction.png|400px]]<br />
<br />
*'''samples'''<br />
#[Plux rep - ''gfp''](BBa_K395101) on pSB6A1 + [PtetR – LuxR] on pSB3K3<br />
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
#negative control: [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3<br />
*'''Strain'''<br />
DH5&alpha;<br />
*'''protocol'''<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan).(→fresh culture)<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.80.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of AHL is 100nM.<br />
#Induction for 2 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
<br />
===M1-2, characterization of K395008 (promoter repressed by LuxR/3OC6HSL):R0061 weak===<br />
We constructed K395105 combining K395008 and K121013. K121013 is a promoter-less ''gfp'' reporter (rbs-''gfp''-ter-ter) and this backbone is pSB6A1. Promoter of S03119 is PtetR, which is repressed by tetR. In this experiment, we don’t use TetR, so, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.<br />
We used a fusion of PlacI<sup>q</sup> (I14032) to ''gfp'' (K121013) as a positive control and used promoterless ''gfp'' (K121013) as a negative control.<br />
<br />
[[IMAGE:Tokyotech_K395008assay_construction.png|400px]]<br />
<br />
*samples<br />
#[R0061weak - ''gfp''](BBa_K395105) on pSB6A1 + [PtetR – LuxR] on pSB3K3<br />
#positive control: [PlacI<sup>q</sup>(constitutive promoter) - ''gfp''] on pSB6A1+ [PtetR – LuxR]) on pSB3K3 <br />
#negative control: [promoterless - ''gfp''] on pSB6A1+ [PtetR – LuxR] on pSB3K3 <br />
*Strain<br />
DH5&alpha;<br />
*protocol<br />
#Prepare overnight culture.<br />
#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)<br />
#Incubate the fresh culture until the observed O.D. reaches around 0.60.<br />
#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.<br />
#Induction for 3 hours at 37°C.<br />
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.<br />
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=Reference=<br />
#KRISTI A. EGLAND & E. P. GREENBERG, Conversion of the Vibrio fischeri Transcriptional Activator LuxR, to a Repressor. JOURNAL OF BACTERIOLOGY, Feb. 2000, p. 805–811<br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assayTeam:Tokyo Tech/Project/Artificial Cooperation System/Cm assay2010-10-28T03:15:51Z<p>Kitano: /* The growth assay */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:3-2 resistance gene activation device -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3-2 resistance gene activation device</b></font><br />
==Abstract==<br />
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (''CmR'') gene ([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by ''lux'' activation 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 />
[[Image:tokyotech_Cm-survival.jpg|thumb|fig.3-3-1|400px|left]]<br />
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==Introduction==<br />
Chloramphenicol is a well known antibiotic that is effective against a wide variety of Gram-positive and Gram-negative bacteria including ''E. coli''. Chloramphenicol stops bacterial growth because it is a protein synthesis inhibitor, which prevents peptide bond formation. When chloramphenicol is added, the synthesis of proteins is inhibited in bacteria which don't express chloramphenicol resistance gene. Then their growth stops and the number of the cell is going to decrease. However, bacteria which express chloramphenicol resistance gene are able to survive in the existence of chloramphenicol. These characteristics indicates that chloramphenicol and chloramphenicol resistance gene can be used for population control which is important to construct the Artificial Cooperation System<br />
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==Results==<br />
*The results of 20 hours incubation after addition of chloramphenicol shows that P''lux''-CmR grew when 3OC6HSL is added , while P''lux''-CmR in the absence of 3OC6HSL was not able to grow.<br />
On the other hand, The cells introduced the promoterless device were not able to grow independently of 3OC6HSL. In the case of the device that had constitutive promoter, the growth of the cells introduced the device were quite slow compared with that of P''lux''. However, there was no significant difference between in the presence and the absence of 3OC6HSL.<br />
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[[Image:Tokyotech_cm_assay.jpg|600px|thumb|Fig.3-3-2 Growth curve of the cell introduced PlacI<sup>q</sup>-''CmR'' (BBa_K395165,blue curves), P''lux''-''CmR'' (BBa_K395162,Red curves), or promoterless-''CmR'' (BBa_K395160,green curves) with or without AHL (3OC6HSL).<br>This work is done by Yusuke Kaneta.]]<br />
<br />
==Conclusion==<br />
From the results, when chloramphenicol was added, the cells introduced P''lux''-''CmR'' were able to grow because LuxR/3OC6HSL complex activated P''lux''. This means we found the conditions that integrants were able to survive only in the presence of 3OC6HSL.<br />
<br />
==Material and Method==<br />
===construction P''lux''-''CmR'' on pSB6A1===<br />
A part, RBS-''CmR'' was cut from BBa_P1004 by PCR and put into pSB6A1. Next RBS-''CmR'' on pSB6A1 and P''lux'' (BBa_R0062) was cut at EcoRI /Xba site and EcoRI/SpeI site respectively. Then we ligated two parts.<br />
===Construction of ''E. coli'' strain DH5&alpha;===<br />
Competent cells with ''luxR'' gene on pSB3K3 were constructed. Afterward, P''lux''-''CmR'' on pSB6A1 was introduced into the cells.<br />
===The growth assay===<br />
In order to follow up the growth of the cells which P''lux''-''CmR'' was introduced into, the change of O.D. (590nm) in both the presence and the absence of 3OC6HSL was measured.<br><br />
(samples)<br><br />
#P''lux''-''CmR ''[http://partsregistry.org/Part:BBa_K395162 BBa_K395162]<br />
#constitutive promoter (PlacI<sup>q</sup>)-''CmR '' (positive control)[http://partsregistry.org/Part:BBa_K395165 BBa_K395165]<br />
#promoterless-''CmR '' (negative control)[http://partsregistry.org/Part:BBa_K395160 BBa_K395160]<br><br />
<br />
LB medium were used for liquid culture. Antibiotics (ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm)) were dissolved in distillation water and stored as 25 mg/ml, whose final concentration in the medium was 50ug/ml, 30ug/ml, and 750ug/ml respectively.<br />
<br />
#The seed cultures of the samples were inoculated from glycerol stock solution and grown separately overnight at 37°C in LB medium containing Amp & Kan.<br />
#Then, we diluted the cultures 100 folds by two types of 3 ml of fresh medium. One type of the medium contains 3OC6HSL (final concentration:100nM). Another contains DMSO as control. Both of the cultures were incubated at 37°C<br />
#1 hour after dilution, each culture was separated to two new tubes. Then it was added 1.5 ml LB medium each containing Cm (final concentration:750ug/ml), Amp, Kan,3OC6HSL or DMSO<br />
#Measured O.D.590 every an hour, up to 20 hours from dilution.<br />
<br />
==Reference==<br />
#Ying-jin Yuan ''et al'' PLoS 2010, e10619<br />
#W. Shaw, ''et al'' Br. Med. Bull. 1984, 40, 36. <br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assayTeam:Tokyo Tech/Project/Artificial Cooperation System/Cm assay2010-10-28T03:15:23Z<p>Kitano: /* The growth assay */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:3-2 resistance gene activation device -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3-2 resistance gene activation device</b></font><br />
==Abstract==<br />
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (''CmR'') gene ([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by ''lux'' activation 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 />
[[Image:tokyotech_Cm-survival.jpg|thumb|fig.3-3-1|400px|left]]<br />
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<br />
==Introduction==<br />
Chloramphenicol is a well known antibiotic that is effective against a wide variety of Gram-positive and Gram-negative bacteria including ''E. coli''. Chloramphenicol stops bacterial growth because it is a protein synthesis inhibitor, which prevents peptide bond formation. When chloramphenicol is added, the synthesis of proteins is inhibited in bacteria which don't express chloramphenicol resistance gene. Then their growth stops and the number of the cell is going to decrease. However, bacteria which express chloramphenicol resistance gene are able to survive in the existence of chloramphenicol. These characteristics indicates that chloramphenicol and chloramphenicol resistance gene can be used for population control which is important to construct the Artificial Cooperation System<br />
<br />
==Results==<br />
*The results of 20 hours incubation after addition of chloramphenicol shows that P''lux''-CmR grew when 3OC6HSL is added , while P''lux''-CmR in the absence of 3OC6HSL was not able to grow.<br />
On the other hand, The cells introduced the promoterless device were not able to grow independently of 3OC6HSL. In the case of the device that had constitutive promoter, the growth of the cells introduced the device were quite slow compared with that of P''lux''. However, there was no significant difference between in the presence and the absence of 3OC6HSL.<br />
<br><br />
<br />
[[Image:Tokyotech_cm_assay.jpg|600px|thumb|Fig.3-3-2 Growth curve of the cell introduced PlacI<sup>q</sup>-''CmR'' (BBa_K395165,blue curves), P''lux''-''CmR'' (BBa_K395162,Red curves), or promoterless-''CmR'' (BBa_K395160,green curves) with or without AHL (3OC6HSL).<br>This work is done by Yusuke Kaneta.]]<br />
<br />
==Conclusion==<br />
From the results, when chloramphenicol was added, the cells introduced P''lux''-''CmR'' were able to grow because LuxR/3OC6HSL complex activated P''lux''. This means we found the conditions that integrants were able to survive only in the presence of 3OC6HSL.<br />
<br />
==Material and Method==<br />
===construction P''lux''-''CmR'' on pSB6A1===<br />
A part, RBS-''CmR'' was cut from BBa_P1004 by PCR and put into pSB6A1. Next RBS-''CmR'' on pSB6A1 and P''lux'' (BBa_R0062) was cut at EcoRI /Xba site and EcoRI/SpeI site respectively. Then we ligated two parts.<br />
===Construction of ''E. coli'' strain DH5&alpha;===<br />
Competent cells with ''luxR'' gene on pSB3K3 were constructed. Afterward, P''lux''-''CmR'' on pSB6A1 was introduced into the cells.<br />
===The growth assay===<br />
In order to follow up the growth of the cells which P''lux''-''CmR'' was introduced into, the change of O.D. (590nm) in both the presence and the absence of 3OC6HSL was measured.<br><br />
(samples)<br><br />
#P''lux''-''CmR ''[http://partsregistry.org/Part:BBa_K395162 BBa_K395162]<br />
#constitutive promoter (PlacI<sup>q</sup>)-''CmR '' (positive control[http://partsregistry.org/Part:BBa_K395165 ) BBa_K395165]<br />
#promoterless-''CmR '' (negative control[http://partsregistry.org/Part:BBa_K395160 ) BBa_K395160]<br><br />
<br />
LB medium were used for liquid culture. Antibiotics (ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm)) were dissolved in distillation water and stored as 25 mg/ml, whose final concentration in the medium was 50ug/ml, 30ug/ml, and 750ug/ml respectively.<br />
<br />
#The seed cultures of the samples were inoculated from glycerol stock solution and grown separately overnight at 37°C in LB medium containing Amp & Kan.<br />
#Then, we diluted the cultures 100 folds by two types of 3 ml of fresh medium. One type of the medium contains 3OC6HSL (final concentration:100nM). Another contains DMSO as control. Both of the cultures were incubated at 37°C<br />
#1 hour after dilution, each culture was separated to two new tubes. Then it was added 1.5 ml LB medium each containing Cm (final concentration:750ug/ml), Amp, Kan,3OC6HSL or DMSO<br />
#Measured O.D.590 every an hour, up to 20 hours from dilution.<br />
<br />
==Reference==<br />
#Ying-jin Yuan ''et al'' PLoS 2010, e10619<br />
#W. Shaw, ''et al'' Br. Med. Bull. 1984, 40, 36. <br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assayTeam:Tokyo Tech/Project/Artificial Cooperation System/Cm assay2010-10-28T03:14:39Z<p>Kitano: /* The growth assay */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:3-2 resistance gene activation device -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3-2 resistance gene activation device</b></font><br />
==Abstract==<br />
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (''CmR'') gene ([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by ''lux'' activation 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 />
[[Image:tokyotech_Cm-survival.jpg|thumb|fig.3-3-1|400px|left]]<br />
<br />
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<br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Introduction==<br />
Chloramphenicol is a well known antibiotic that is effective against a wide variety of Gram-positive and Gram-negative bacteria including ''E. coli''. Chloramphenicol stops bacterial growth because it is a protein synthesis inhibitor, which prevents peptide bond formation. When chloramphenicol is added, the synthesis of proteins is inhibited in bacteria which don't express chloramphenicol resistance gene. Then their growth stops and the number of the cell is going to decrease. However, bacteria which express chloramphenicol resistance gene are able to survive in the existence of chloramphenicol. These characteristics indicates that chloramphenicol and chloramphenicol resistance gene can be used for population control which is important to construct the Artificial Cooperation System<br />
<br />
==Results==<br />
*The results of 20 hours incubation after addition of chloramphenicol shows that P''lux''-CmR grew when 3OC6HSL is added , while P''lux''-CmR in the absence of 3OC6HSL was not able to grow.<br />
On the other hand, The cells introduced the promoterless device were not able to grow independently of 3OC6HSL. In the case of the device that had constitutive promoter, the growth of the cells introduced the device were quite slow compared with that of P''lux''. However, there was no significant difference between in the presence and the absence of 3OC6HSL.<br />
<br><br />
<br />
[[Image:Tokyotech_cm_assay.jpg|600px|thumb|Fig.3-3-2 Growth curve of the cell introduced PlacI<sup>q</sup>-''CmR'' (BBa_K395165,blue curves), P''lux''-''CmR'' (BBa_K395162,Red curves), or promoterless-''CmR'' (BBa_K395160,green curves) with or without AHL (3OC6HSL).<br>This work is done by Yusuke Kaneta.]]<br />
<br />
==Conclusion==<br />
From the results, when chloramphenicol was added, the cells introduced P''lux''-''CmR'' were able to grow because LuxR/3OC6HSL complex activated P''lux''. This means we found the conditions that integrants were able to survive only in the presence of 3OC6HSL.<br />
<br />
==Material and Method==<br />
===construction P''lux''-''CmR'' on pSB6A1===<br />
A part, RBS-''CmR'' was cut from BBa_P1004 by PCR and put into pSB6A1. Next RBS-''CmR'' on pSB6A1 and P''lux'' (BBa_R0062) was cut at EcoRI /Xba site and EcoRI/SpeI site respectively. Then we ligated two parts.<br />
===Construction of ''E. coli'' strain DH5&alpha;===<br />
Competent cells with ''luxR'' gene on pSB3K3 were constructed. Afterward, P''lux''-''CmR'' on pSB6A1 was introduced into the cells.<br />
===The growth assay===<br />
In order to follow up the growth of the cells which P''lux''-''CmR'' was introduced into, the change of O.D. (590nm) in both the presence and the absence of 3OC6HSL was measured.<br><br />
(samples)<br><br />
#P''lux''-''CmR ''[http://partsregistry.org/Part:BBa_K395162 BBa_K395162]<br />
#constitutive promoter (PlacI<sup>q</sup>)-''CmR '' (positive control)[http://partsregistry.org/Part:BBa_K395165 BBa_K395165]<br />
#promoterless-''CmR '' (negative control)[http://partsregistry.org/Part:BBa_K395160 BBa_K395160]<br><br />
<br />
LB medium were used for liquid culture. Antibiotics (ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm)) were dissolved in distillation water and stored as 25 mg/ml, whose final concentration in the medium was 50ug/ml, 30ug/ml, and 750ug/ml respectively.<br />
<br />
#The seed cultures of the samples were inoculated from glycerol stock solution and grown separately overnight at 37°C in LB medium containing Amp & Kan.<br />
#Then, we diluted the cultures 100 folds by two types of 3 ml of fresh medium. One type of the medium contains 3OC6HSL (final concentration:100nM). Another contains DMSO as control. Both of the cultures were incubated at 37°C<br />
#1 hour after dilution, each culture was separated to two new tubes. Then it was added 1.5 ml LB medium each containing Cm (final concentration:750ug/ml), Amp, Kan,3OC6HSL or DMSO<br />
#Measured O.D.590 every an hour, up to 20 hours from dilution.<br />
<br />
==Reference==<br />
#Ying-jin Yuan ''et al'' PLoS 2010, e10619<br />
#W. Shaw, ''et al'' Br. Med. Bull. 1984, 40, 36. <br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assayTeam:Tokyo Tech/Project/Artificial Cooperation System/Cm assay2010-10-28T03:14:03Z<p>Kitano: /* The growth assay */</p>
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:3-2 resistance gene activation device -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3-2 resistance gene activation device</b></font><br />
==Abstract==<br />
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (''CmR'') gene ([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by ''lux'' activation 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 />
[[Image:tokyotech_Cm-survival.jpg|thumb|fig.3-3-1|400px|left]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Introduction==<br />
Chloramphenicol is a well known antibiotic that is effective against a wide variety of Gram-positive and Gram-negative bacteria including ''E. coli''. Chloramphenicol stops bacterial growth because it is a protein synthesis inhibitor, which prevents peptide bond formation. When chloramphenicol is added, the synthesis of proteins is inhibited in bacteria which don't express chloramphenicol resistance gene. Then their growth stops and the number of the cell is going to decrease. However, bacteria which express chloramphenicol resistance gene are able to survive in the existence of chloramphenicol. These characteristics indicates that chloramphenicol and chloramphenicol resistance gene can be used for population control which is important to construct the Artificial Cooperation System<br />
<br />
==Results==<br />
*The results of 20 hours incubation after addition of chloramphenicol shows that P''lux''-CmR grew when 3OC6HSL is added , while P''lux''-CmR in the absence of 3OC6HSL was not able to grow.<br />
On the other hand, The cells introduced the promoterless device were not able to grow independently of 3OC6HSL. In the case of the device that had constitutive promoter, the growth of the cells introduced the device were quite slow compared with that of P''lux''. However, there was no significant difference between in the presence and the absence of 3OC6HSL.<br />
<br><br />
<br />
[[Image:Tokyotech_cm_assay.jpg|600px|thumb|Fig.3-3-2 Growth curve of the cell introduced PlacI<sup>q</sup>-''CmR'' (BBa_K395165,blue curves), P''lux''-''CmR'' (BBa_K395162,Red curves), or promoterless-''CmR'' (BBa_K395160,green curves) with or without AHL (3OC6HSL).<br>This work is done by Yusuke Kaneta.]]<br />
<br />
==Conclusion==<br />
From the results, when chloramphenicol was added, the cells introduced P''lux''-''CmR'' were able to grow because LuxR/3OC6HSL complex activated P''lux''. This means we found the conditions that integrants were able to survive only in the presence of 3OC6HSL.<br />
<br />
==Material and Method==<br />
===construction P''lux''-''CmR'' on pSB6A1===<br />
A part, RBS-''CmR'' was cut from BBa_P1004 by PCR and put into pSB6A1. Next RBS-''CmR'' on pSB6A1 and P''lux'' (BBa_R0062) was cut at EcoRI /Xba site and EcoRI/SpeI site respectively. Then we ligated two parts.<br />
===Construction of ''E. coli'' strain DH5&alpha;===<br />
Competent cells with ''luxR'' gene on pSB3K3 were constructed. Afterward, P''lux''-''CmR'' on pSB6A1 was introduced into the cells.<br />
===The growth assay===<br />
In order to follow up the growth of the cells which P''lux''-''CmR'' was introduced into, the change of O.D. (590nm) in both the presence and the absence of 3OC6HSL was measured.<br><br />
(samples)<br><br />
#P''lux''-''CmR ''[http://partsregistry.org/Part:BBa_K395162 BBa_K395162]<br />
#constitutive promoter (PlacI<sup>q</sup>)-''CmR'' (positive control)[http://partsregistry.org/Part:BBa_K395165 BBa_K395165]<br />
#promoterless-''CmR'' (negative control)[http://partsregistry.org/Part:BBa_K395160 BBa_K395160]<br><br />
<br />
LB medium were used for liquid culture. Antibiotics (ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm)) were dissolved in distillation water and stored as 25 mg/ml, whose final concentration in the medium was 50ug/ml, 30ug/ml, and 750ug/ml respectively.<br />
<br />
#The seed cultures of the samples were inoculated from glycerol stock solution and grown separately overnight at 37°C in LB medium containing Amp & Kan.<br />
#Then, we diluted the cultures 100 folds by two types of 3 ml of fresh medium. One type of the medium contains 3OC6HSL (final concentration:100nM). Another contains DMSO as control. Both of the cultures were incubated at 37°C<br />
#1 hour after dilution, each culture was separated to two new tubes. Then it was added 1.5 ml LB medium each containing Cm (final concentration:750ug/ml), Amp, Kan,3OC6HSL or DMSO<br />
#Measured O.D.590 every an hour, up to 20 hours from dilution.<br />
<br />
==Reference==<br />
#Ying-jin Yuan ''et al'' PLoS 2010, e10619<br />
#W. Shaw, ''et al'' Br. Med. Bull. 1984, 40, 36. <br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assayTeam:Tokyo Tech/Project/Artificial Cooperation System/Cm assay2010-10-28T03:11:13Z<p>Kitano: /* The growth assay */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:3-2 resistance gene activation device -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3-2 resistance gene activation device</b></font><br />
==Abstract==<br />
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (''CmR'') gene ([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by ''lux'' activation 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 />
[[Image:tokyotech_Cm-survival.jpg|thumb|fig.3-3-1|400px|left]]<br />
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==Introduction==<br />
Chloramphenicol is a well known antibiotic that is effective against a wide variety of Gram-positive and Gram-negative bacteria including ''E. coli''. Chloramphenicol stops bacterial growth because it is a protein synthesis inhibitor, which prevents peptide bond formation. When chloramphenicol is added, the synthesis of proteins is inhibited in bacteria which don't express chloramphenicol resistance gene. Then their growth stops and the number of the cell is going to decrease. However, bacteria which express chloramphenicol resistance gene are able to survive in the existence of chloramphenicol. These characteristics indicates that chloramphenicol and chloramphenicol resistance gene can be used for population control which is important to construct the Artificial Cooperation System<br />
<br />
==Results==<br />
*The results of 20 hours incubation after addition of chloramphenicol shows that P''lux''-CmR grew when 3OC6HSL is added , while P''lux''-CmR in the absence of 3OC6HSL was not able to grow.<br />
On the other hand, The cells introduced the promoterless device were not able to grow independently of 3OC6HSL. In the case of the device that had constitutive promoter, the growth of the cells introduced the device were quite slow compared with that of P''lux''. However, there was no significant difference between in the presence and the absence of 3OC6HSL.<br />
<br><br />
<br />
[[Image:Tokyotech_cm_assay.jpg|600px|thumb|Fig.3-3-2 Growth curve of the cell introduced PlacI<sup>q</sup>-''CmR'' (BBa_K395165,blue curves), P''lux''-''CmR'' (BBa_K395162,Red curves), or promoterless-''CmR'' (BBa_K395160,green curves) with or without AHL (3OC6HSL).<br>This work is done by Yusuke Kaneta.]]<br />
<br />
==Conclusion==<br />
From the results, when chloramphenicol was added, the cells introduced P''lux''-''CmR'' were able to grow because LuxR/3OC6HSL complex activated P''lux''. This means we found the conditions that integrants were able to survive only in the presence of 3OC6HSL.<br />
<br />
==Material and Method==<br />
===construction P''lux''-''CmR'' on pSB6A1===<br />
A part, RBS-''CmR'' was cut from BBa_P1004 by PCR and put into pSB6A1. Next RBS-''CmR'' on pSB6A1 and P''lux'' (BBa_R0062) was cut at EcoRI /Xba site and EcoRI/SpeI site respectively. Then we ligated two parts.<br />
===Construction of ''E. coli'' strain DH5&alpha;===<br />
Competent cells with ''luxR'' gene on pSB3K3 were constructed. Afterward, P''lux''-''CmR'' on pSB6A1 was introduced into the cells.<br />
===The growth assay===<br />
In order to follow up the growth of the cells which P''lux''-''CmR'' was introduced into, the change of O.D. (590nm) in both the presence and the absence of 3OC6HSL was measured.<br><br />
(samples)<br><br />
#P''lux''-''CmR ''[http://partsregistry.org/Part:BBa_K395162 BBa_K395162]<br />
#constitutive promoter (PlacI<sup>q</sup>)-''CmR'' (positive control)[http://partsregistry.org/Part:BBa_K395165 BBa_K395165]<br />
#promoterless-''CmR'' (negative control)[http://partsregistry.org/Part:BBa_K395160 BBa_K395160]<br><br />
<br />
LB medium were used for liquid culture. Antibiotics (ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm)) were dissolved in distillation water and stored as 25 mg/ml, whose final concentration in the medium was 50ug/ml, 30ug/ml, and 750ug/ml respectively.<br />
<br />
#The seed cultures of the samples were inoculated from glycerol stock solution and grown separately overnight at 37°C in LB medium containing Amp & Kan.<br />
#Then, we diluted the cultures 100 folds by two types of 3 ml of fresh medium. One type of the medium contains 3OC6HSL (final concentration:100nM). Another contains DMSO as control. Both of the cultures were incubated at 37°C<br />
#After 1 hour from dilution, each culture was separated to two new tubes. Then it was added 1.5 ml LB medium each containing Cm (final concentration:750ug/ml), Amp, Kan,3OC6HSL or DMSO<br />
#Measured O.D.590 every an hour, up to 20 hours from dilution.<br />
<br />
==Reference==<br />
#Ying-jin Yuan ''et al'' PLoS 2010, e10619<br />
#W. Shaw, ''et al'' Br. Med. Bull. 1984, 40, 36. <br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
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<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assayTeam:Tokyo Tech/Project/Artificial Cooperation System/Cm assay2010-10-28T03:08:29Z<p>Kitano: /* The growth assay */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:3-2 resistance gene activation device -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
</th><br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3-2 resistance gene activation device</b></font><br />
==Abstract==<br />
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (''CmR'') gene ([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by ''lux'' activation 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 />
[[Image:tokyotech_Cm-survival.jpg|thumb|fig.3-3-1|400px|left]]<br />
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<br />
==Introduction==<br />
Chloramphenicol is a well known antibiotic that is effective against a wide variety of Gram-positive and Gram-negative bacteria including ''E. coli''. Chloramphenicol stops bacterial growth because it is a protein synthesis inhibitor, which prevents peptide bond formation. When chloramphenicol is added, the synthesis of proteins is inhibited in bacteria which don't express chloramphenicol resistance gene. Then their growth stops and the number of the cell is going to decrease. However, bacteria which express chloramphenicol resistance gene are able to survive in the existence of chloramphenicol. These characteristics indicates that chloramphenicol and chloramphenicol resistance gene can be used for population control which is important to construct the Artificial Cooperation System<br />
<br />
==Results==<br />
*The results of 20 hours incubation after addition of chloramphenicol shows that P''lux''-CmR grew when 3OC6HSL is added , while P''lux''-CmR in the absence of 3OC6HSL was not able to grow.<br />
On the other hand, The cells introduced the promoterless device were not able to grow independently of 3OC6HSL. In the case of the device that had constitutive promoter, the growth of the cells introduced the device were quite slow compared with that of P''lux''. However, there was no significant difference between in the presence and the absence of 3OC6HSL.<br />
<br><br />
<br />
[[Image:Tokyotech_cm_assay.jpg|600px|thumb|Fig.3-3-2 Growth curve of the cell introduced PlacI<sup>q</sup>-''CmR'' (BBa_K395165,blue curves), P''lux''-''CmR'' (BBa_K395162,Red curves), or promoterless-''CmR'' (BBa_K395160,green curves) with or without AHL (3OC6HSL).<br>This work is done by Yusuke Kaneta.]]<br />
<br />
==Conclusion==<br />
From the results, when chloramphenicol was added, the cells introduced P''lux''-''CmR'' were able to grow because LuxR/3OC6HSL complex activated P''lux''. This means we found the conditions that integrants were able to survive only in the presence of 3OC6HSL.<br />
<br />
==Material and Method==<br />
===construction P''lux''-''CmR'' on pSB6A1===<br />
A part, RBS-''CmR'' was cut from BBa_P1004 by PCR and put into pSB6A1. Next RBS-''CmR'' on pSB6A1 and P''lux'' (BBa_R0062) was cut at EcoRI /Xba site and EcoRI/SpeI site respectively. Then we ligated two parts.<br />
===Construction of ''E. coli'' strain DH5&alpha;===<br />
Competent cells with ''luxR'' gene on pSB3K3 were constructed. Afterward, P''lux''-''CmR'' on pSB6A1 was introduced into the cells.<br />
===The growth assay===<br />
In order to follow up the growth of the cells which P''lux''-''CmR'' was introduced into, the change of O.D. (590nm) in both the presence and the absence of 3OC6HSL was measured.<br><br />
(samples)<br><br />
#P''lux''-''CmR ''[http://partsregistry.org/Part:BBa_K395162 BBa_K395162]<br />
#constitutive promoter (PlacI<sup>q</sup>)-''CmR'' (positive control)[http://partsregistry.org/Part:BBa_K395165 BBa_K395165]<br />
#promoterless-''CmR'' (negative control)[http://partsregistry.org/Part:BBa_K395160 BBa_K395160]<br><br />
<br />
LB medium were used for liquid culture. Antibiotics (ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm)) were dissolved in distillation water and stored as 25 mg/ml, whose final concentration in the medium was 50ug/ml, 30ug/ml, and 750ug/ml respectively.<br />
<br />
#The seed cultures of the samples were inoculated from glycerol stock solution and grown separately overnight at 37°C in LB medium containing Amp&Kan.<br />
#Then, we diluted the cultures 100 folds by two types of 3 ml of fresh medium. One type of the medium contains 3OC6HSL (final concentration:100nM). Another contains DMSO as control. Both of the cultures were incubated at 37°C<br />
#After 1 hour from dilution, each culture was separated to two new tubes. Then it was added 1.5 ml LB medium each containing Cm (final concentration:750ug/ml), Amp, Kan,3OC6HSL or DMSO<br />
#Measured O.D.590 every an hour, up to 20 hours from dilution.<br />
<br />
==Reference==<br />
#Ying-jin Yuan ''et al'' PLoS 2010, e10619<br />
#W. Shaw, ''et al'' Br. Med. Bull. 1984, 40, 36. <br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assayTeam:Tokyo Tech/Project/Artificial Cooperation System/Cm assay2010-10-28T03:06:46Z<p>Kitano: /* Results */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:3-2 resistance gene activation device -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
</th><br />
</tr><br />
<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3-2 resistance gene activation device</b></font><br />
==Abstract==<br />
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (''CmR'') gene ([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by ''lux'' activation 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 />
[[Image:tokyotech_Cm-survival.jpg|thumb|fig.3-3-1|400px|left]]<br />
<br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Introduction==<br />
Chloramphenicol is a well known antibiotic that is effective against a wide variety of Gram-positive and Gram-negative bacteria including ''E. coli''. Chloramphenicol stops bacterial growth because it is a protein synthesis inhibitor, which prevents peptide bond formation. When chloramphenicol is added, the synthesis of proteins is inhibited in bacteria which don't express chloramphenicol resistance gene. Then their growth stops and the number of the cell is going to decrease. However, bacteria which express chloramphenicol resistance gene are able to survive in the existence of chloramphenicol. These characteristics indicates that chloramphenicol and chloramphenicol resistance gene can be used for population control which is important to construct the Artificial Cooperation System<br />
<br />
==Results==<br />
*The results of 20 hours incubation after addition of chloramphenicol shows that P''lux''-CmR grew when 3OC6HSL is added , while P''lux''-CmR in the absence of 3OC6HSL was not able to grow.<br />
On the other hand, The cells introduced the promoterless device were not able to grow independently of 3OC6HSL. In the case of the device that had constitutive promoter, the growth of the cells introduced the device were quite slow compared with that of P''lux''. However, there was no significant difference between in the presence and the absence of 3OC6HSL.<br />
<br><br />
<br />
[[Image:Tokyotech_cm_assay.jpg|600px|thumb|Fig.3-3-2 Growth curve of the cell introduced PlacI<sup>q</sup>-''CmR'' (BBa_K395165,blue curves), P''lux''-''CmR'' (BBa_K395162,Red curves), or promoterless-''CmR'' (BBa_K395160,green curves) with or without AHL (3OC6HSL).<br>This work is done by Yusuke Kaneta.]]<br />
<br />
==Conclusion==<br />
From the results, when chloramphenicol was added, the cells introduced P''lux''-''CmR'' were able to grow because LuxR/3OC6HSL complex activated P''lux''. This means we found the conditions that integrants were able to survive only in the presence of 3OC6HSL.<br />
<br />
==Material and Method==<br />
===construction P''lux''-''CmR'' on pSB6A1===<br />
A part, RBS-''CmR'' was cut from BBa_P1004 by PCR and put into pSB6A1. Next RBS-''CmR'' on pSB6A1 and P''lux'' (BBa_R0062) was cut at EcoRI /Xba site and EcoRI/SpeI site respectively. Then we ligated two parts.<br />
===Construction of ''E. coli'' strain DH5&alpha;===<br />
Competent cells with ''luxR'' gene on pSB3K3 were constructed. Afterward, P''lux''-''CmR'' on pSB6A1 was introduced into the cells.<br />
===The growth assay===<br />
In order to follow up the growth of the cells which P''lux''-''CmR'' was introduced into, the change of O.D. (590nm) in both the presence and the absence of 3OC6HSL was measured.<br><br />
(samples)<br><br />
#P''lux''-''CmR''[http://partsregistry.org/Part:BBa_K395162 BBa_K395162]<br />
#constitutive promoter (PlacI<sup>q</sup>)-''CmR'' (positive control)[http://partsregistry.org/Part:BBa_K395165 BBa_K395165]<br />
#promoterless-''CmR'' (negative control)[http://partsregistry.org/Part:BBa_K395160 BBa_K395160]<br><br />
<br />
LB medium were used for liquid culture. Antibiotics (ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm)) were dissolved in distillation water and stored as 25 mg/ml, whose final concentration in the medium was 50ug/ml, 30ug/ml, and 750ug/ml respectively.<br />
<br />
#The seed cultures of the samples were inoculated from glycerol stock solution and grown separately overnight at 37°C in LB medium containing Amp&Kan.<br />
#Then, we diluted the cultures 100 folds by two types of 3 ml of fresh medium. One type of the medium contains 3OC6HSL (final concentration:100nM). Another contains DMSO as control. Both of the cultures were incubated at 37°C<br />
#After 1 hour from dilution, each culture was separated to two new tubes. Then it was added 1.5 ml LB medium each containing Cm (final concentration:750ug/ml), Amp, Kan,3OC6HSL or DMSO<br />
#Measured O.D.590 every an hour, up to 20 hours from dilution.<br />
<br />
==Reference==<br />
#Ying-jin Yuan ''et al'' PLoS 2010, e10619<br />
#W. Shaw, ''et al'' Br. Med. Bull. 1984, 40, 36. <br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assayTeam:Tokyo Tech/Project/Artificial Cooperation System/Cm assay2010-10-28T03:04:34Z<p>Kitano: /* Results */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
<tr><br />
<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:3-2 resistance gene activation device -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
</th><br />
</tr><br />
<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3-2 resistance gene activation device</b></font><br />
==Abstract==<br />
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (''CmR'') gene ([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by ''lux'' activation 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 />
[[Image:tokyotech_Cm-survival.jpg|thumb|fig.3-3-1|400px|left]]<br />
<br />
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<br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Introduction==<br />
Chloramphenicol is a well known antibiotic that is effective against a wide variety of Gram-positive and Gram-negative bacteria including ''E. coli''. Chloramphenicol stops bacterial growth because it is a protein synthesis inhibitor, which prevents peptide bond formation. When chloramphenicol is added, the synthesis of proteins is inhibited in bacteria which don't express chloramphenicol resistance gene. Then their growth stops and the number of the cell is going to decrease. However, bacteria which express chloramphenicol resistance gene are able to survive in the existence of chloramphenicol. These characteristics indicates that chloramphenicol and chloramphenicol resistance gene can be used for population control which is important to construct the Artificial Cooperation System<br />
<br />
==Results==<br />
*The results of 20 hours incubation after addition of chloramphenicol shows that P''lux''-CmR grew when 3OC6HSL is added , while P''lux''-CmR in the absence of 3OC6HSL was not able to grow.<br />
On the otherhand, The cells introduced the promoterless device were not able to grow independently of 3OC6HSL. In the case of the device that had constitutive promoter, the growth of the cells introduced the device were quite slow compared with that of P''lux''. However, there was no significant difference between in the presence and the absent of 3OC6HSL.<br />
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[[Image:Tokyotech_cm_assay.jpg|600px|thumb|Fig.3-3-2 Growth curve of the cell introduced PlacI<sup>q</sup>-''CmR'' (BBa_K395165,blue curves), P''lux''-''CmR'' (BBa_K395162,Red curves), or promoterless-''CmR'' (BBa_K395160,green curves) with or without AHL (3OC6HSL).<br>This work is done by Yusuke Kaneta.]]<br />
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==Conclusion==<br />
From the results, when chloramphenicol was added, the cells introduced P''lux''-''CmR'' were able to grow because LuxR/3OC6HSL complex activated P''lux''. This means we found the conditions that integrants were able to survive only in the presence of 3OC6HSL.<br />
<br />
==Material and Method==<br />
===construction P''lux''-''CmR'' on pSB6A1===<br />
A part, RBS-''CmR'' was cut from BBa_P1004 by PCR and put into pSB6A1. Next RBS-''CmR'' on pSB6A1 and P''lux'' (BBa_R0062) was cut at EcoRI /Xba site and EcoRI/SpeI site respectively. Then we ligated two parts.<br />
===Construction of ''E. coli'' strain DH5&alpha;===<br />
Competent cells with ''luxR'' gene on pSB3K3 were constructed. Afterward, P''lux''-''CmR'' on pSB6A1 was introduced into the cells.<br />
===The growth assay===<br />
In order to follow up the growth of the cells which P''lux''-''CmR'' was introduced into, the change of O.D. (590nm) in both the presence and the absence of 3OC6HSL was measured.<br><br />
(samples)<br><br />
#P''lux''-''CmR''[http://partsregistry.org/Part:BBa_K395162 BBa_K395162]<br />
#constitutive promoter (PlacI<sup>q</sup>)-''CmR'' (positive control)[http://partsregistry.org/Part:BBa_K395165 BBa_K395165]<br />
#promoterless-''CmR'' (negative control)[http://partsregistry.org/Part:BBa_K395160 BBa_K395160]<br><br />
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LB medium were used for liquid culture. Antibiotics (ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm)) were dissolved in distillation water and stored as 25 mg/ml, whose final concentration in the medium was 50ug/ml, 30ug/ml, and 750ug/ml respectively.<br />
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#The seed cultures of the samples were inoculated from glycerol stock solution and grown separately overnight at 37°C in LB medium containing Amp&Kan.<br />
#Then, we diluted the cultures 100 folds by two types of 3 ml of fresh medium. One type of the medium contains 3OC6HSL (final concentration:100nM). Another contains DMSO as control. Both of the cultures were incubated at 37°C<br />
#After 1 hour from dilution, each culture was separated to two new tubes. Then it was added 1.5 ml LB medium each containing Cm (final concentration:750ug/ml), Amp, Kan,3OC6HSL or DMSO<br />
#Measured O.D.590 every an hour, up to 20 hours from dilution.<br />
<br />
==Reference==<br />
#Ying-jin Yuan ''et al'' PLoS 2010, e10619<br />
#W. Shaw, ''et al'' Br. Med. Bull. 1984, 40, 36. <br />
<!--ここまで書いて良いですよ--><br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assayTeam:Tokyo Tech/Project/Artificial Cooperation System/Cm assay2010-10-28T03:01:42Z<p>Kitano: /* Introduction */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:3-2 resistance gene activation device -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
</th><br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3-2 resistance gene activation device</b></font><br />
==Abstract==<br />
We succeeded in constructing and characterizing a NEW Biobrick device of chloramphenicol resistance (''CmR'') gene ([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by ''lux'' activation 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 />
[[Image:tokyotech_Cm-survival.jpg|thumb|fig.3-3-1|400px|left]]<br />
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==Introduction==<br />
Chloramphenicol is a well known antibiotic that is effective against a wide variety of Gram-positive and Gram-negative bacteria including ''E. coli''. Chloramphenicol stops bacterial growth because it is a protein synthesis inhibitor, which prevents peptide bond formation. When chloramphenicol is added, the synthesis of proteins is inhibited in bacteria which don't express chloramphenicol resistance gene. Then their growth stops and the number of the cell is going to decrease. However, bacteria which express chloramphenicol resistance gene are able to survive in the existence of chloramphenicol. These characteristics indicates that chloramphenicol and chloramphenicol resistance gene can be used for population control which is important to construct the Artificial Cooperation System<br />
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==Results==<br />
*The results of 20 hours incubation after addition of chloramphenicol shows that P''lux''-CmR grew when 3OC6HSL is added ,while P''lux''-CmR in the absence of 3OC6HSL was not able to grow.<br />
On the otherhand, The cells introduced the promoterless device were not able to grow independently of 3OC6HSL. In the case of the device that had constitutive promoter, the growth of the cells introduced the device were quite slow compared with that of P''lux''. However, there was no significant difference between in the presence and the absent of 3OC6HSL.<br />
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[[Image:Tokyotech_cm_assay.jpg|600px|thumb|Fig.3-3-2 Growth curve of the cell introduced PlacI<sup>q</sup>-''CmR'' (BBa_K395165,blue curves), P''lux''-''CmR'' (BBa_K395162,Red curves), or promoterless-''CmR'' (BBa_K395160,green curves) with or without AHL (3OC6HSL).<br>This work is done by Yusuke Kaneta.]]<br />
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==Conclusion==<br />
From the results, when chloramphenicol was added, the cells introduced P''lux''-''CmR'' were able to grow because LuxR/3OC6HSL complex activated P''lux''. This means we found the conditions that integrants were able to survive only in the presence of 3OC6HSL.<br />
<br />
==Material and Method==<br />
===construction P''lux''-''CmR'' on pSB6A1===<br />
A part, RBS-''CmR'' was cut from BBa_P1004 by PCR and put into pSB6A1. Next RBS-''CmR'' on pSB6A1 and P''lux'' (BBa_R0062) was cut at EcoRI /Xba site and EcoRI/SpeI site respectively. Then we ligated two parts.<br />
===Construction of ''E. coli'' strain DH5&alpha;===<br />
Competent cells with ''luxR'' gene on pSB3K3 were constructed. Afterward, P''lux''-''CmR'' on pSB6A1 was introduced into the cells.<br />
===The growth assay===<br />
In order to follow up the growth of the cells which P''lux''-''CmR'' was introduced into, the change of O.D. (590nm) in both the presence and the absence of 3OC6HSL was measured.<br><br />
(samples)<br><br />
#P''lux''-''CmR''[http://partsregistry.org/Part:BBa_K395162 BBa_K395162]<br />
#constitutive promoter (PlacI<sup>q</sup>)-''CmR'' (positive control)[http://partsregistry.org/Part:BBa_K395165 BBa_K395165]<br />
#promoterless-''CmR'' (negative control)[http://partsregistry.org/Part:BBa_K395160 BBa_K395160]<br><br />
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LB medium were used for liquid culture. Antibiotics (ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm)) were dissolved in distillation water and stored as 25 mg/ml, whose final concentration in the medium was 50ug/ml, 30ug/ml, and 750ug/ml respectively.<br />
<br />
#The seed cultures of the samples were inoculated from glycerol stock solution and grown separately overnight at 37°C in LB medium containing Amp&Kan.<br />
#Then, we diluted the cultures 100 folds by two types of 3 ml of fresh medium. One type of the medium contains 3OC6HSL (final concentration:100nM). Another contains DMSO as control. Both of the cultures were incubated at 37°C<br />
#After 1 hour from dilution, each culture was separated to two new tubes. Then it was added 1.5 ml LB medium each containing Cm (final concentration:750ug/ml), Amp, Kan,3OC6HSL or DMSO<br />
#Measured O.D.590 every an hour, up to 20 hours from dilution.<br />
<br />
==Reference==<br />
#Ying-jin Yuan ''et al'' PLoS 2010, e10619<br />
#W. Shaw, ''et al'' Br. Med. Bull. 1984, 40, 36. <br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompCTeam:Tokyo Tech/Project/wolf coli/New Series of PompC2010-10-28T02:55:31Z<p>Kitano: /* New BioBrick Parts */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<td>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
</td><br />
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<tr><br />
<th>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:4-1 New seriesof P''ompC'' &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>4-1 New series of P''ompC''</b></font><br />
==Abstract==<br />
In order to fine tune the Wolf coli 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.<br />
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==New BioBrick Parts==<br />
[[Image:Tokyotech_ompc_graph.jpg|left|thumb|300px|Fig.4-1-1 The induction of new P''OmpC'' series in high osmolarity medium at 4 hours. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA Tokyo Tech iGEM 2010]] <br />
[[Image:Tokyotech wolfcoli system_ver5.png|thumb|right|300px|Fig. 4-1-2. Overview of “Wolfcoli” system]]<br />
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(Fig. 4-1-1) We have succeeded in designing 2 new osmoregulative promoters, P''OmpC(CB)'' and P''OmpC(CS1)'', which can also be utilized in red-light-dependent gene expression network (Fig.4-1-2).<br />
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==Introduction==<br />
*;What is ''OmpC'' promoter?<br />
[[image:Tokyotech_OmpC_wild_type.jpg|center|400px|PompC(WT) and the two-component system]]<br />
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''OmpC(WT)'' promoter (BBa_R0082) is positively regulated by phosphorylated OmpR, a response regulator in EnvZ-OmpR osmosensing two-component system. The EnvZ-OmpR system of E. coli regulates the transcriptional activity of this promoter in response to change in extracellular osmolarity. The ''OmpC(WT)'' promoter consists of 3 distinct phosphorylated OmpR recognition sites which are binding site 1, binding site 2 and binding site 3.<br />
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==Result==<br />
*;'''The new series of ''OmpC'' promoters'''<br />
[[image:Tokyotech_New_pompC_construction.jpg|center|400px|PompC(WT) and the two-component system]]<br />
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Three new promoters, P''ompC(CS1)'' BBa_395303, P''ompC(C)'' BBa_395301, P''ompC(CB)'' BBa_395302 were designed based on previous paper[1]. The second promoter, P''ompC(CS1)'', comprises only binding site1 fused to the canonical -35 and -10 region. P''ompC(C)'' is a composite of binding site 1 and additional 4 base pairs before the canonical -35 and -10 region. Likewise, P''ompC(CB)'' consists of binding site1 and the additional 10 base pairs before the -35 and -10 consensus sequence.<br />
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*; Characterization of the new series of ''OmpC'' promoters<br />
[[Image:Tokyotech_ompc_graph.jpg|thumb|left|400px|Fig. 4-1-1 The induction of new'' OmpC'' series in high osmolarity medium at 4 hours. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA ]] <br />
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After 4 hours of sucrose induction, transcriptional activity of PompC(CB)-GFP and PompC(CS1)-GFP increased 2.5-folds and 2.3-folds respectively. However, significant amount of leaky expression was found in PompC(CS1)-GFP without induction. In contrast, under the same conditions, we found no significant difference of GFP expression in PompC(C)-GFP. Although, PompC(WT)-GFP also doesn't show difference in expression of GFP at 4 hours of induction, there was slightly higher GFP expression 1.7-folds occurred in the wild type at 2 hours.<br />
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==Discussion==<br />
The transcriptional activity of PompC(WT)-GFP increased in particular period and declined after it reached the peak of expression. This result shows the relation to the study by the [http://partsregistry.org/Part:BBa_R0082:Experience Edinburgh iGEM 2009]. Moreover, we considereated that we can create varities of P''OmpC'' by combining OmpR binding sites on the upstream of the ''OmpC'' promoters with spacer sequence.<br />
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==Conclusion==<br />
According to the results, we have succeeded in comprising 2 new series of osmoregulative promoters. Furthermore, we expect that P''ompC(CB)'' would be a key component in responding to the middle light intensitys, “full moon light” and probably awaken our werewolf coli in the “full moon night”<br />
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==Material and Method==<br />
===Construction of E. coli strain MG1655=== <br />
Each new BioBrick part, P''ompC(C)''-GFP [http://partsregistry.org/Part:BBa_K395304 BBa_K395304 ], P''ompC(CB)''-GFP [http://partsregistry.org/Part:BBa_K395305 BBa_K395305 ], P''ompC(CS1)''-GFP [http://partsregistry.org/Part:BBa_K395306 BBa_K395306 ] and P''ompC(WT)''-GFP [http://partsregistry.org/Part:BBa_K395307 BBa_K395307 ] on pSB3K3 was introduced into E. coli strain MG1655. A strain containing plac<sup>q</sup>-GFP plasmid (BBa_J54202), a constitutive GFP expressive promoter, and promoterless GFP reporter plasmid (BBa_J54103) was used as a positive control and negative control respectively.<br />
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===Medium===<br />
Medium A, per liter, contains 7 g of nutrient broth, 1 g of yeast extract, 2 g of glycerol, 3.7 g of K2HPO4, and 1.3 g of KH2PO4 [3].<br />
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===Report assay===<br />
In order to determine the strength of promoter parts in the reporter plasmid, the change of fluorescence intensities of the reporter stains in the presence and the absence of the inducer were measured. Overnight cultures of reporter strains grown at 37 °C in Medium A containing appropriated antibiotics were diluted at least 1:100 in the medium and incubated at 37 °C as fresh cultures. After their OD<sub>590</sub> reached 0.2, the fresh culture was diluted 1 : 3 into 2 ml of pre-warmed medium A. Under high osmolarity conditions, the cultures were diluted with sucrose supplemented medium to the final concentration of 15% (wt/vol)[2].<br />
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===Sample preparation for GFP measurement===<br />
After 4 hours of high ormolarity induction, 0.2 ml of each culture was moved to 2.0 ml eppendorf tube and then centrifuged for 1 min at 4°C, 9000 rpm. The supernatant was discarded from each tube by pipette. In order to adjust each sample to reach approximately same final OD<sub>590</sub>, appropriate amount of 1×PBS washing solution was added. The cell pellet in each tube was resuspended by vortexing. After all samples for GFP measurement were prepared, 150 &mu;l of each sample was applied to 96-well plate and its fluorescence intensity was measured with fluorometer. The fluorescence intensity was calculated by dividing the measured raw fluorescence intensity by the adjusted OD<sub>590</sub>.<br />
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==References==<br />
1. Maeda S. & Mizuno T. Activation of the Osmoregulated ''ompC'' Gene by the ''OmpR'' Protein in Escherichia coli. J.Biochem 1991;110,324-27 <br><br />
2. Batchelor E. & Goulian M. Imaging ''OmpR'' localization in Escherichia coli. Molecular Microbiology. 2006;59(6),1767-78 <br><br />
3. Kawaji H. Influence of Molecular Size and Osmolarity of Sugars and Dextrans on the Synthesis of Outer Membrane Proteins 0-8 and 0-9 of Escherichia coli K-12. J.Bacteriology 1979;140(3), 843-47<br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/lacIM1Team:Tokyo Tech/Project/wolf coli/lacIM12010-10-28T02:39:44Z<p>Kitano: /* Construction of E.coli strain DH5α */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
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<td>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<th>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:4-2 lacIM1 for band-detect network&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>4-2 lacIM1 for band-detect network</b></font><br />
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<!--ここから書き始めて下さい--><br />
==Abstract==<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) [3], 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.<br />
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(Fig. 4-2-1).<br />
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[[Image:Tokyotech LacIM1 system ver4.png|left|thumb|300px|Figure4-2-1. ]]<br />
[[Image:Tokyotech_LacIM1_data_ver2.png|right|thumb|300px|Figure4-2-2. Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG. This work is done by Mitsuhiko Odera ]]<br />
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==Introduction==<br />
===What is LacIM1?===<br />
As I wrote before, LacIM1 shows weaker repression compare to LacIWT because of its lower affinity to lac promoter [1].<br />
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===Requirement for band-detect network===<br />
The band-detect network exhibits transient gene expression in response to concentration of chemical signals. In the band-detect network, LacIM1 is a key component because its repression efficiency can afford the network the desired non-monotonic response.<br />
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==Results==<br />
[[Image: Tokyotech_LacIM1_data_ver2.png|left|thumb|300px|Figure4-2-2. Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG. This work is done by Mitsuhiko Odera ]]<br />
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(Figure4-2-2. <br />
Repression efficiency of LacIM1 ([http://partsregistry.org/Part:BBa_K395401 BBa_K395401]) / LacIWT ([http://partsregistry.org/Part:BBa_K395402 BBa_K395402]) exposed to arabinose and IPTG.<br><br />
Dark-blue bars, 0.2% arabinose and 0 mM IPTG;<br><br />
blue bars, 0% arabinose and 0 mM IPTG; <br><br />
dark-green bars, 0.2% arabinose and 1 mM IPTG;<br />
green bars, 0% arabinose and 1 mM IPTG;<br><br />
red bar, positive control<br><br />
orange bar, negative control <br><br />
GFP expression is dependent on the input of arabinose and IPTG. After 4 hours of arabinose and IPTG induction. Cultures were analysed by fluorometer (FLA5000、FUJI).<br />
As positive control, we used BBa_I20260, which is measurement kit of the iGEM.)<br />
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After 4 hours of arabinose and IPTG induction. Cultures were analysed by fluorometer (FLA5000, FUJI).<br />
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This is the result of LacIM1. <br><br />
In the absence of both arabinose and IPTG, GFP expression is about 1.8-fold stronger than that of the presence of arabinose and the absence of IPTG. We confirmed the dependency of GFP expression on product of lacIM1 by arabinose induction. In other words, the fact that LacIM1 represses the GFP expression was corroborated, based on our results. Moreover, Increase of GFP production by addition of IPTG, with or without arabinose induction, again shows that the repressions are dependent on LacIM1. Without arabinose induction, increase of GFP production by addition of IPTG, can be explained due to the leaky expression of LacIM1. This leaky expression could also explain the results for LacWT.<br />
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This is the result of LacIWT.<br><br />
・In the presence of arabinose and the absence of IPTG, GFP expression did not occur. This results show that LacI protein was expressed by arabinose induction and could fully repressed GFP expression.<br><br />
Absence of both arabinose and IPTG. Normally without arabinose induction, the GFP is supposed to be expressed. However, GFP expression did not occur. This result shows that the leaky expression of LacI protein is been sufficient for repressing GFP expression.<br><br />
・Presence of both arabinose and IPTG. Normally, although arabinose induces the overproduction of LacI protein and represses the GFP expression, LacI protein can be inhibited by IPTG. Therefore, we expected the occurrence of GFP expression. However, there is no expression of GFP. This result shows that the amount of IPTG was not sufficient to inhibit the repression by overproduced LacI proteins. <br><br />
・In the absence of arabinose and the presence of IPTG, as I mentioned before, leaky expression of LacI protein occurs. However, when there is only small amount of LacI protein, addition of IPTG can fully inhibit the repression of LacI protein. Thus, GFP expression occurs.<br />
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==Conclusion==<br />
We confirmed that lacIM1 shows weaker repression compare to LacIWT.<br />
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==Materials&Methods==<br />
===Construction of ''E.coli'' strain DH5α===<br />
In order to characterize K082026(K395400), LacI Mutant, we constructed BBa_K395401 combining I0500 and K082026(K395400) on pSB1A3. and used BBa_I20260 as a positive control and used promoterless gfp on pSB3K3 as a negative control.<br />
Furthermore, we constructed BBa_K395402 combining I0500 and BBa_I 732820 on pSB1A3 as a control experiment.<br />
As a GFP reporter, we used BBa_I7106 on pSB3K3. <br />
In order to measure the function of lacI proteins, we introduced BBa_I7106 (lacI+pL-rbs-GFP-ter) on pSB3K3 and BBa_K395401/BBa_K395402 into DH5α.<br />
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===Report assay=== <br />
Overnight cultures of reporter strains grown at 37 °C in LB Medium containing appropriated antibiotics were diluted at least 1:100 in the medium and were incubated at 37 °C as fresh cultures. After their OD590 reached 0.3, added appropriate inducers that 1mM IPTG, and/or 0.2% arabinose (final concentration). <br />
After 4 hours of induction, 150 l of each sample was applied to 96-well plate and its fluorescence intensity was measured with a fluorometer by BPB-sp filter, which emit LB. The fluorescence intensity was calculated by dividing the measured raw fluorescence intensity by the adjusted OD590. <br />
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==Refference==<br />
[1]. Basu S, Gerchman Y, Collins CH, et al. A synthetic multicellular system for programmed pattern formation. NATURE 2005;434,1130-1134 <br><br />
[2]. USTC(2008)<br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coliTeam:Tokyo Tech/Project/wolf coli2010-10-28T02:38:55Z<p>Kitano: /* Introduction */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<td>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<th>4 Wolf coli overview -YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>4 Wolf coli Overview</b></font><br />
==Introduction==<br />
[[Image:tokyotech_wolfcoli_system_ver2.png|left|thumb|250px|Fig. 4-1 Cooperative activity of Artificial Cooperative System]]<br />
[[Image:tokyotech_wolfcoli_system_ver5.png|right|thumb|350px|Figure 4-2. Overview of “Wolf coli” system]]<br />
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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"[1] and "band-detect network"[2] into one cell, and combined these networks with the Artificial Cooperation System (Fig. 4-3). We characterized new series of ''OmpC'' promoter, and LacIM1 which are crucial parts of our networks.<br />
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[[Image:Tokyotech_ompc_graph.jpg|thumb|left|320px|Fig. 4-3 The induction of new P''ompC'' series in high osmolarity medium at 4 hours. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA]]<br />
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[[Image:Tokyotech_LacIM1_data.png|thumb|right|230px|Fig. 4-4 Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG. This work is done by Mitsuhiko ODERA]]<br />
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We have succeeded in designing 2 new osmoregulative promoters, P''ompC(CB)'' [http://partsregistry.org/Part:BBa_K395302 (BBa_K395302)] and P''ompC(CS1)''[http://partsregistry.org/Part:BBa_K395303 (BBa_K395303)], which can be utilized in the red-light-dependent gene expression network (Fig. 4-3). We also characterized LacIM1 [http://partsregistry.org/Part:BBa_K082026 (BBa_K082026)], a mutant of LacIWT,which is a key component in the band-detect network. Although, this part was registered by USTC(2008) [3], it was not well characterized in the BioBrick registry. We confirmed that LacIM1 shows weaker repression to Plac than its wild type. (Fig. 4-4)<br />
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==Wolf coli. and Artificial Cooperative System ==<br />
[[image:Tokyotech_Cooperative_activity.jpg|center|300px]]<br />
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The Aftificial Cooperation System was designed to be switching off during the “full moon night”. Therefore, Sympathetic coli would be transformed into Wolf coli at the “full moon light”. During this period, communication between 2 types of cells is inhibited. Hence, both types of cells are unable to recognize each other by quorum sensing and become competitors. Cooperative activity in Artificial Cooperation System is regulated by 3 levels of light intensity which are weak, medium and strong. “Weak light” from the crescent moon enables the Artificial Cooperation System on, thus two types of cell are able to communicate and help each other. “Medium light” during the full moon night can switch the system off resulting in appearing of the “Wolf coli”. During the daytime, “strong light” from the sun activates the quorum sensing resulting in turning on of the Aftificial Cooperation System.<br />
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==P''OmpC'' in red-light-dependent gene expression network==<br />
[[Image:Tokyotech wolfcoli system_ver5.png|left|thumb|300px|Fig. 4-5 Overview of red-light-dependent gene expression network in Wolf coli system]]<br />
[[Image:Tokyotech_ompc_graph.jpg|thumb|left|320px|Fig. 4-6 The induction of new P''ompC'' series in high osmolarity medium at hours. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA]]<br />
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''OmpC'' promoter, in red-light-dependent gene expression network, plays a crucial role in initiating the transformation of Sympathetic coli into Werewolf coli. To accomplish band-detect circuit in light sensening system, varieties of ''OmpC'' promoters were designed and characterized so as to find an appropriate strength of the promoter particularly which can be activated by the “full moon light”.(Fig.4-5)<br />
[https://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC#Introduction ...see more about ''OmpC'' promoter ](Fig. 4-6)<br />
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==LacIMI in band-detect network==<br />
[[image:Tokyotech_band_detect.png|340px|thumb|left| Fig.4-7 Patial circuit of band-detect network ]]<br />
[[Image:Tokyotech_LacIM1_data.png|thumb|right|240px|Fig. 4-8.Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG. This work is done by Mitsuhiko ODERA]]<br />
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The band-detect network exhibits transient gene expression in response to concentration of chemical signals(Fig.4-7). In the band-detect network, LacIM1 is a crucial component due to its low repression efficiency[1]. According to the assay results, we confirmed that LacIM1 shows weaker repression to Plac than its wild type.(Fig. 4-8). [https://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/lacIM1 …see more about lacIM1]. <br />
==References==<br />
[1]. Basu S, Gerchman Y, Collins CH, et al. A synthetic multicellular system for programmed pattern formation. NATURE 2005;434,1130-1134 <br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter2Team:Tokyo Tech/Project/Apple Reporter22010-10-28T02:35:54Z<p>Kitano: /* Result */</p>
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<table id="table-01"><br />
<tr><br />
<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<th>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:2-2 Fragrance -YOU ARE HERE!-<br />
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<td>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>2-2 Fragrance</b></font><br />
=Abstract=<br />
We designed apple fragrance expression device with MpAAT1<sup>[1]</sup>. MpAAT1 is able to produce ester compounds with apple fragrance using alcohols and Acetyl-CoA. Fig. 2-2-1 shows the outline of the device. We performed gas chromatography to confirm the product of esters. The results revealed that MpAAT1 could produce 2-methylbutyl acetate and butyl acetate using 2-methyl butanol and butanol respectively (Fig. 2-2-2). 2-methylbutyl acetate and butyl acetate are known as the Apple fragrance.<br />
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[[image:Tokyotech apple fragrance device.png|630px|center|thumb|Fig. 2-2-1. Apple fragrance expression device. <Br>Abbreviations : 2-methyl butanol (2-MeBuOH), butanol (BuOH), 2-methylbutyl acetate (2-MeBuOAc), butyl acetate (BuOAc).]]<br />
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[[image:Tokyotech_gas_chromatograpy.png|630px|center|thumb| Fig. 2-2-2. Gas chromatography analysis of apple fragrance expression device. <br>(1) MpAAT1 + BuOH, (2) No MpAAT1 + BuOAc, (3) No MpAAT1 + BuOH, (4) MpAAT1 + 2-MeBuOH, (5) No MpAAT1 + 2-MeBuOAc, (6) No MpAAT1 + 2-MeBuOH, (7) MpAAT1 - 2-MeBuOH.<br>This work is done by Toshitaka Matsubara.]]<br />
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=Introduction=<br />
''MpAAT1'' gene was isolated from ''Malus pumila'' (popular apple)<sup>[2]</sup>. This gene is expressed in leaves, flowers and fruit of apple. The recombinant enzyme (MpAAT1) to produce esters involved in apple fragrance, utilize various alcohol as substrates such as straight chain (C3–C10), branched chain, aromatic and terpene alcohols. In addition, various kinds of CoA derivatives, such as acetyl-CoA, are used for producing esters by MpAAT1. Both alcohol and CoA derivative are required for successful ester synthesis. The major components involved in apple fragrance are butyl acetate and 2-methylbutyl acetate<sup>[3]</sup>. We engineered apple fragrance expression device to produce these molecules by MpAAT1. This device is used to Artificial Cooperation System in our projects, when dying cells are recued by its counterpart, it gives an apple fragrance for a token of their gratitude.<br />
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=Result=<br />
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We transformed MpAAT1([http://partsregistry.org/Part:BBa_K395602 BBa_K395602]) on pSB6A1 along with pTrx6 into ''E.coli'' BL21 DE3, and cultured after addition of alcohols(2-MeBuOH or BuOH) as substrates.<br />
After 12 hours of incubation, we extracted organic solution layer from the culture and analyzed by gas chromatography (Fig. 2-2-2). Peaks of the esters producing apple fragrance (2-MeBuOAc or BuOAc) were detected.<br />
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[[image:Tokyotech_gas_chromatograpy.png|630px|left|thumb|Fig. 2-2-2. Gas chromatography analysis of apple fragrance expression device. <br>(1) MpAAT1 + BuOH, (2) No MpAAT1 + BuOAc, (3) No MpAAT1 + BuOH, (4) MpAAT1 + 2-MeBuOH, (5) No MpAAT1 + 2-MeBuOAc, (6) No MpAAT1 + 2-MeBuOH, (7) MpAAT1 - 2-MeBuOH.<br />
<br>This work is done by Toshitaka Matsubara.]]<br />
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By comparing (1) with (2), BuOAc was confirmed when ''E. coli'' has MpAAT1. Moreover, by Comparing (1) with (3), BuOH (substrate) doesn’t contain BuOAc as impurity. By the same token, 2-MeBuOAc was confirmed when ''E. coli'' has MpAAT1 by comparing (4) with (5). Moreover, by Comparing (4) with (6), 2-MeBuOH (substrate) doesn’t contain 2-MeBuOAc as impurity. (7) shows ''E. coli'' which has MpAAT1 is able to convert alcohols (substrates) into fragrance molecules grown in no substrate culture. From these result, ''E. coli'' which has MpAAT1 successfully was able to produce 2-methylbutyl acetate and butyl acetate using 2-methyl butanol and butanol respectively.<br />
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=Discussion=<br />
From the result of the experiment above, we can conclude that apple fragrance expression device was able to produce esters using alcohols added as substrates.<br />
Moreover, we synthesized MpAAT1 and esters using ''E.coli'' BL21 DE3 as a chassis, which was reported to be implausible in former report(s) <sup>[1]</sup>.<br />
In 2008, C.R. Shen and J.C. Liao<sup>[4]</sup> succeeded in synthesizing butanol from ''E.coli''. If we take advantage of this engineered ''E.coli'', we could produce apple fragrance ester without the addition of substrate.<br />
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=Materials and Methods=<br />
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'''Strains of ''E. coli'''''<br />
<Br>''E. coli'' BL21 DE3<br />
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'''Varieties of plasmid'''<Br><br />
MpAAT1 on pSB6A1<Br><br />
Trx on pACYC184<br />
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'''Substrate'''<Br><br />
Butanol (final 0.4%)<Br><br />
2-methyl butanol (final 0.2%)<Br><br />
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'''Inducer'''<Br><br />
100 mM IPTG<Br><br />
20% arabinose<Br><br />
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'''Solution'''<Br><br />
Undecane solution: undecane 10 &mu;L + ether 990 &mu;L<br />
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'''MpAAT1 expression construct. '''<Br><br />
We ordered the synthesis of ''MpAAT1''([http://partsregistry.org/Part:BBa_K395602 BBa_K395602]) from Mr. GENE.<Br><br />
This artificial gene was annealed with vector pSB6A1 as MpAAT1 expression plasmid.<Br><br />
Moreover, we introduced pTrx6 into this expression plasmid to stabilize the ''MpAAT1'' gene product.<Br><br />
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MpAAT1 over expression conditions<Br><br />
Artificial gene has T7 promoter on the upstream of ''MpAAT1''.<Br><br />
This promoter works by taking over T7 RNA polymerase from ''E. coli''.<Br><br />
Therefore we utilized ''E. coli'' BL21 DE3 which has T7 RNA polymerase.<Br><br />
Furthermore, arabinose was added in culture to induce Trx which has arabinose-induced promoter.<Br><br />
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''E. coli'' BL21 DE3 <Br><br />
''E. coli'' BL21 DE3 express T7 RNA polymerase by IPTG induction.<Br><br />
Therefore we added 3 &mu;L of 100 mM IPTG and 15 &mu;L of 20% arabinose in LB culture in order to express MpAAT1 and Trx in ''E. coli'' BL21 DE3.<Br><br />
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Expression of MpAAT1 recombinant protein in ''E. coli''<Br><br />
O/N ''E. coli''<Br><br />
→100-fold dilution <Br><br />
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→make up the OD<sub>590</sub> = 0.1 with LB culture<Br><br />
→add substrate,antibiotics and inducer<Br><br />
→O/N<Br><br />
→harvest (7,000×''g'', 3 min)<Br><br />
→collect supernatant solution<Br><br />
→separating liquid layer and oil layer with ether (shake supernatant solution 0.5 mL, ether 0.5 mL and undecane soln. 2 &mu;L)<Br><br />
→derive oil layer from supernatant solution with liquid-liquid extraction<Br><br />
→GC analysis.<Br><br />
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GC analysis<Br><br />
GC: SHIMADZU GAS CHROMATOGRAPH GC-14B<Br><br />
Column: J&W SCIENTIFIC, DB-17, Film thickness 0.25 &mu;m, Column Dimensions 15 m × 0.320 mm, Temperature Limits 40°C to 280°C (300°C Program)<Br><br />
Conditions: column temperature 35°C, injector temperature 180°C, detector temperature 180°C<Br><br />
Sample was injected 5 &mu;L.<Br><br />
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=reference=<br />
[1] [[media:リンゴのにおい.pdf|Edwige J. F. Souleyre, FEBS Journal, 272, 3132–3144 (2005)]]<Br><br />
[2] [[media:リンゴ元.pdf|Young H, ''J Sci Food Agric'', 71, 329-336 (1996)]]<Br><br />
[3] [http://www.ncbi.nlm.nih.gov/nucleotide/52139952 GenBank accession number AY707098]<Br><br />
[4] [[media:Utanol.pdf|C.R. Shen, Metabolic Engineering, 10, 312–320 (2008)]]<Br><br />
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</div> <!-- end Super_main_wrapper --</div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Apple_ReporterTeam:Tokyo Tech/Project/Apple Reporter2010-10-28T02:29:13Z<p>Kitano: /* gathering E. coli and extract pigment */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<th>2 Apple reporter<br><br />
:2-1 Color -YOU ARE HERE!-<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<td>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New series of P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>2-1 Color</b></font><br />
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==Abstract==<br />
Our team synthesized &beta;-carotene, zeaxanthin and astaxanthin under several conditions (strain, plasmid copy number,…etc) to confirm the functions. Particularly, '''astaxanthin is the final metabolite of the carotenoid synthetic pathway.''' <br />
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We synthesized &beta;-carotene and zeaxanthin by assembling ''crtEBIY'' and ''crtZ'' of existing BioBrick parts. '''Though the part ''crtZ'' has been unconfirmed to produce zeaxanthin, we confirmed it for the first time in iGEM.''' <br />
<br />
In addition, we succeeded in synthesizing astaxanthin by introducing '''the new BioBrick part ''crtW'''''. Therefore, the carotenoid synthetic pathway reached the end.<br />
<br />
<br />
[[Image:tokyotech_Fig. 1.1.1. carotenoid synthetic pathway.jpg|390px|left|thumb|Fig. 2-1-1. carotenoid synthetic pathway]]<br />
<br />
[[Image:TLC work3.jpeg|right|210px|thumb|Fig.2-1-2.TLC worked by Yumiko Kinoshita]]<br />
<br />
<br />
<br />
[https://2010.igem.org/Team:Tokyo_Tech/Parts &rarr;our BioBrick parts]<br />
[http://partsregistry.org/Part:BBa_K395704 &rarr;BBa_395704]<br />
[http://partsregistry.org/Part:BBa_K395706 &rarr;BBa_395706]<br />
<br />
==Introduction==<br />
Carotenoids are natural organic pigments in plants and bacteria. Synthetic carotenoid pigments colored yellow, red or orange represent about 15-25% of the cost of production of commercial feed. Carotenoids are terpenoid based on a structure having the chemical formula C<sub>40</sub>H<sub>56</sub>. There are about 600 carotenoids, which perform a wide range of functions. (For example, acting as antioxidants[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Reference], and as precursors to other organic compounds.) '''Among carotenoids, astaxanthin is not known to be converted to Vitamin A, which is converted from most of carotenoids and is toxic when its concentration too high.''' <br />
<br />
Previous year, our team has completed melanin synthetic pathway. Since the carotenoid synthetic pathway is huge, BioBrick Registry doesn’t cover all parts to complete the pathway. '''This year, we introduced a new BioBrick part to synthesize more various kinds of carotenoids. And we succeeded in synthesizing astaxanthin, one of the final metabolite of the pathway.''' <br />
<br />
[[Image:tokyotech_tokyotech_Fig. 1.1.5. note.jpg|500px|center|thumb|Fig. 2-1-3. note]]<br />
<br />
==Results==<br />
<br />
===&beta;-carotene synthesis===<br />
[[Image:tokyotech_Fig. 1.1.2.1. &beta;-carotene.jpg|right|200px|thumb|Fig.2-1-4.&beta;-carotene]]<br />
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Our team synthesized &beta;-carotene under several conditions to confirm the functions of BioBrick parts designed by Cambridge 2009. (we call it "crtEBIY plasmids".) ([https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#appendix See more...])<br />
<br />
===zeaxanthin synthesis===<br />
[[Image:tokyotech_Fig. 1.1.6. zeaxanthin.jpg|right|200px|thumb|Fig.2-1-5zeaxanthin]]<br />
Our team tried to synthesize zeaxanthin by the construct ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395704 BBa_K395704]) to confirm the activity of BioBrick parts designed by Cambredge 2009 and Edinburgh 2007. We also confirmed zeaxanthin by TLC (Fig.2-1-6 spot III). <br />
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<br />
By introduction of ''crtZ'' gene to the ''crtEBIY'' plasmid (we call this construct “single plasmid construct”, "''crtZ-crtEBIY''".), new spot appered on TLC plate(Fig.2-1-6 spot III). The spot shows lower Rf value than that derived from ''crtEBIY'' (Fig.2-1-6 spot II). The Rf values of spots delived from ''crtEBIY'' and ''crtZ-crtEBIY'' corresponded to reported values of &beta;-caroten and Zeaxanthin, respectively.[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Reference] Thus we could characterize zeaxanthin production activity of crtZ. In addition, caracterization of the part ''crtZ'' is the first work in iGEM. <br />
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In order to maximize zeaxanthin production, we designed some combinations of construct. We thought of another type of zeaxanthin synthetic construct where ''“crtZ”'' (BBa_I742158) and ''“crtEBIY”'' (BBa_K274210) were separated on different plasmids. (we call this type of constructs “double plasmids construct.”)<br />
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After all, We made five combinations shown in table<1>, including [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395701 BBa_K395701], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395702 BBa_K395702] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395704 BBa_K395704].<br />
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Constructs containing pBad/araC promoter on high-copy vector show the best production and double plasmids construct shows as much production as single plasmid construct under same promoter combination. Construct with pBad/araC promoter shows better production than that with plac promoter. Construct that ''crtEBIY'' on high-copy vector and ''crtZ'' on low-copy vector shows as much expression as construct that ''crtEBIY'' on low-copy vector and ''crtZ'' on high-copy vector.(Actually, #3 showed a little more production than #5.) <br />
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For TLC, We selected the best three samples including the sample derived from single plasmid construct . <br />
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In the consequence of TLC, we could confirm that these three samples were zeaxanthin.<br />
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[[Image:tokyotech_Fig. 1.1.7.1.BBa_K395704.jpg|left|300px|thumb|S1]]<br />
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[[Image:TLC work3.jpeg|right|300px|thumb|Fig.2-1-6 zeaxanthin TLC worked by Yumiko Kinoshita]]<br />
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[[Image:Tokyotech_zeaxanthin_synthesis.jpg|center|500px|thumb|Fig.2-1-7 zeaxanthin worked by Yumiko Kinoshita]]<br />
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===astaxanthin synthesis===<br />
[[Image:tokyotech_Fig. 1.1.9. zeaxanthin.jpg|right|200px|thumb|Fig.2-1-8 astaxanthin]]<br />
Our team synthesized astaxanthin for the first time in iGEM. Thus the carotenoid synthetic pathway reached the end. This work was done by assembling the new BioBrick part ''crtW'' and zeaxanthin synthesis constructs we designed. From the result of “zeaxanthin synthesis”, we decided to use pBad/araC promoter and double plasmids construct.<br />
After constructing the new BioBrick device ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395706 BBa_K395706]) to synthesize astaxanthin, we characterize the function under the condition I in table<2>. Thus we confirmed astaxanthin by TLC spot IV. <br />
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By introduction of ''crtW'' gene to the ''crtZ'' plasmid (the second construct of five combinations in “zeaxanthin synthesis”,having pBad/araC promoter), new spot (IV) appered on TLC plate. The spot shows lower Rf value than that derived from ''crtEBIY'' (II) and a little higher Rf value than that derived from ''crtZ-crtW''(III). All the Rf values of spots corresponded to reported values respectively. Thus we could characterize astaxanthin production activity of ''crtW''. ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K395705 BBa_K395705], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K395706 BBa_K395706]) <br />
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For the same reason as “zeaxanthin synthesis”, we examined under several conditions &rarr;table<2>.<br />
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In the consequence of TLC, we could confirm that these four samples were astaxanthin.<br />
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Not only astaxanthin but also another spots were produced at II. <br />
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We regarded them as intermediary metabolites between zeaxanthin and astaxanthin.<br />
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The activity of pBad/araC promoter is measured in detail on another page. [https://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/lacIM1 &rarr;click]<br />
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<br />
[[Image:tokyotech_Fig. 2-1-10 1BBa_K395706.jpg|left|300px|thumb|S2]]<br />
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[[Image:TLC work3.jpeg|right|300px|thumb|Fig.2-1-9 TLC astaxanthin worked by Yumiko Kinoshita]]<br />
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[[Image:Tokyotech_astaxanthin_synthesis.jpg|center|500px|thumb|Fig.2-1-10 astaxanthin worked by Yumiko Kinoshita]]<br />
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acetone extract and vacuum concentration [https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Materials_and_Methods &rarr;protocol]<br />
<br />
==Reference==<br />
[1] Yousry M. Antioxidant Activities of Astaxanthin and Related Carotenoids. J. Agric. Food Chem. 2000, 48, 1150-1154<br />
[2]N.M. Sachindra, et al. Carotenoids in crabs from marine and fresh waters of India. LWT 38 (2005) 221–225<br />
<br />
[3]MISAWA N, et al. Structure and Functional Analysis of a Marine Bacterial Carotenoid Biosynthesis Gene Cluster and Astaxanthin Biosynthetic Pathway Proposed at the Gene Level. JOURNAL OF BACTERIOLOGY, Nov. 1995, p. 6575–6584 Vol. 177, No. 22<br />
<br />
[4]Seon-Kang C, et al. Characterization of bacterial β-carotene Ketolases, CrtW,<br />
from Mrine Bacteria by Complementation Analysis in ''Escherichia coli''. MARINE BIOTECHNOLOGY(2005) 7, 515-522<br />
<br />
[5]Seon-Kang C, et al. Characterization of bacterial β-carotene 3,3′-hydroxylases, CrtZ,<br />
and P450 in astaxanthin biosynthetic pathway and adonirubin<br />
production by gene combination in ''Escherichia coli''. Appl Microbiol Biotechnol (2006) 72<br />
<br />
<br />
[6]Nishizaki T, et al. Metabolic engineering of carotenoid biosynthesis in ''Escherichia coli'' by ordered gene assembly in Bacillus subtilis. Appl Environ Microbiol. 2007 Feb<br />
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[7]Arunkumar K, et al. Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase(crtZ)gene by functional complementation in ''Escherichia coli''. Carotenoid Science Vol.11 (2007)<br />
<br />
==appendix==<br />
===&beta;-carotene synthesis===<br />
[[Image:tokyotech_Fig. 1.1.2.1. &beta;-carotene.jpg|right|200px|thumb|Fig.2-1-4 &beta;-carotene]]<br />
Our team synthesized &beta;-carotene under several conditions to confirm the activity of BioBrick parts designed by Cambredge 2009. <br />
<br />
We used ''crtEBIY'' (BBa_K274210) to synthesize &beta;-carotene. We compared the plasmids containing pSB3K3 (low-copy vector) with the plasmids containing pSB1A2 (high-copy vector) from the production. We also introduced the low-copy plasmid into ''E. Coli'' strain MG1655, JM109 and DH5&alpha; respectively. <br />
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We found that MG1655 and JM109 produce more &beta;-carotene than DH5α in liquid culture and that MG1655 grow better than JM109 on plate culture. These results indicated that MG1655 is the best of the three strains to treat. For this reason, we decided to use only MG1655 to extract pigment.<br />
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We also made special competent cell, MG1655 and JM109, having a plasmid ''crtEBIY''; pSB1A2 or ''crtEBIY''; pSB3K3) for zeaxanthin and astaxanthin synthesis.(→[https://2010.igem.org/Image:Tokyotech_Fig._1.1.7.1.BBa_K395704.jpg S1], [https://2010.igem.org/Image:Tokyotech_Fig._2-1-10_1BBa_K395706.jpg S2])<br />
<br />
<br />
~acetone extract~<br />
[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Materials_and_Methods &rarr;protocol]<br />
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[[Image:tokyotech_Fig. 1.1.3. &beta;-carotene extract.jpg|300px|left|thumb|Fig.2-1-11. &beta;-carotene extract /worked by Yumiko Kinoshita]]<br />
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[[Image:tokyotech_Fig. 1.1.4. &beta;-carotene plate.jpg|300px|right|thumb|Fig.2-1-12. &beta;-carotene plate /worked by Yumiko Kinoshita]]<br />
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[[Image:Βカロテン2.png|200px|center|thumb|Fig.2-1-13. &beta;-carotene table /worked by Yumiko Kinoshita]]<br />
<br />
==Materials and Methods==<br />
===The work flow to TLC from culture[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Reference],[https://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter#Reference]===<br />
====Material====<br />
''E. coli'' : strain MG1655 <br />
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reagent : acetone, methanol, chloroform, hexane<br />
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TLC plate : silicagel 60F<sub>254</sub><br />
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====culture====<br />
1. The cells were cultured over night.<br />
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2. 50ul bacterial sution was taken from culture 1 and moved into 50ml LB culture with appropriate antibiotic. Inducer was added, if necessary.<br />
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3. The cells were incubated at 37°C, 24hour.<br />
<br />
====gathering ''E. coli'' and extract pigment====<br />
4. After the O.D reach 2.0, culture was centrifuged for 10minutes at <br />
4°C, 6000rpm,10min<br />
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5. Supernatant was removed by decanting. The remainder was added with 1 ml ddH<sub>2</sub>0 and vortexed.<br />
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6. Moved into 2ml tube by using P1000 pipetter at least twice.<br />
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7. Solution is centrifuged for 20minutes at 4℃, 14000rpm<br />
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8. Supernatant was removed completely using pipetter. About 100ul of pellet is obtained.<br />
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9. Pellet is added with 500ul acetone, and vortexed for 10 minutes to extract the pigment.<br />
10. Solution is centrifuged for 20minutes at 4℃, 14000rpm<br />
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11. Acetone solution was replaced into 2 ml tube.<br />
<br />
====vacuum and concentrate====<br />
12. Acetone was removed using rotary evaporator.<br />
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13. 50 ul of Chloroform/Methanol solution(weight ratio of 9:1) was added.<br />
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14. Sample was vortexed and spinned down.<br />
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15. The water layer was removed.<br />
<br />
====TLC====<br />
16. Spot the sample onto TLC silica gel plate, and developed by acetone/hexane solution.(weight ratio of 1:3)<br />
<br />
17. Spot was observed under visible light.</div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-28T02:25:37Z<p>Kitano: /* Conclusion */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
<br />
=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to activate chloramphenicol resistance gene. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. In cell A (Fig 3-0-2), production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B (Fig3-0-3), production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. On the other hand, 3OC12HSL activates resistance gene of cell B and 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
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<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
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<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062, both of which are existing parts. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), GFP expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, GFP expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|left|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, ''E. coli'' with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-28T00:21:38Z<p>Kitano: /* Model development */</p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
</td><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
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[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
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==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|Table1]]. <br />
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[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
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In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
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'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
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[[IMAGE:Tokyotech S9.jpg|400px]],<br />
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where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
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When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
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[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
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The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
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The growth inhibition by antibiotics is represented by the following equations:<br />
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[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
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where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
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The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
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[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
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Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
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[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
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'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
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[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
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Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
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[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
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where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
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'''III, AHL synthesis and degradation'''<br />
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AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
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[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
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where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
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'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
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[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
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Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
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<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
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Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
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==Prameters==<br />
The parameters used in equation S1 to S8 are listed in Table1. Many parameter values are taken from references. However, quantitative information is not sufficient for some parameters; therefore we use educated guesses that are biologically feasible. <br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
1, Bo Hu, Jin Du, Rui-yang Zou, Ying-jin Yuan, An Environment-Sensitive Synthetic Microbial Ecosystem, PLoS ONE, volume 5 issue 5, 2010<br><br />
2, Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187.<br><br />
3, Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.<br><br />
4, Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional<br />
communication mediates a consensus in a microbial biofilm consortium. Proc<br />
Natl Acad Sci USA 104: 17300–17304.<br><br />
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<!--ここまで書いて良いですよ--><br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-28T00:18:31Z<p>Kitano: </p>
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<tr><br />
<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
<tr><br />
<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
</tr><br />
<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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</table><br />
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<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
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[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
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==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
<br />
[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
<br />
In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
<br />
<br />
'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
<br />
<br />
[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
<br />
When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
<br />
[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
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The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
<br />
The growth inhibition by antibiotics is represented by the following equations:<br />
<br />
[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
<br />
where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
<br />
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The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
<br />
[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
<br />
Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
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[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
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'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
<br />
[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
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Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
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[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
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where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
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'''III, AHL synthesis and degradation'''<br />
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AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
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[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
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where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
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'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
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[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
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Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
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<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
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Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
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==Prameters==<br />
The parameters used in equation S1 to S8 are listed in Table1. Many parameter values are taken from references. However, quantitative information is not sufficient for some parameters; therefore we use educated guesses that are biologically feasible. <br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
<br />
==References==<br />
1, Bo Hu, Jin Du, Rui-yang Zou, Ying-jin Yuan, An Environment-Sensitive Synthetic Microbial Ecosystem, PLoS ONE, volume 5 issue 5, 2010<br><br />
2, Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187.<br><br />
3, Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.<br><br />
4, Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional<br />
communication mediates a consensus in a microbial biofilm consortium. Proc<br />
Natl Acad Sci USA 104: 17300–17304.<br><br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-28T00:17:50Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
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[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
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==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
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[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
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In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
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'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
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[[IMAGE:Tokyotech S9.jpg|400px]],<br />
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where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
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When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
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[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
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The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
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The growth inhibition by antibiotics is represented by the following equations:<br />
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[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
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where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
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The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
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[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
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Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
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[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
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'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
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[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
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Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
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[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
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where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
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'''III, AHL synthesis and degradation'''<br />
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AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
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[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
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where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
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'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
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[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
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Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
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<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
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Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
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==Prameters==<br />
The parameters used in equation S1 to S8 are listed in Table1. Many parameter values are taken from references. However, quantitative information is not sufficient for some parameters; therefore we use educated guesses that are biologically feasible. <br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
1, Bo Hu, Jin Du, Rui-yang Zou, Ying-jin Yuan, An Environment-Sensitive Synthetic Microbial Ecosystem, PLoS ONE, volume 5 issue 5, 2010<br><br />
2, Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187.<br><br />
3, Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.<br><br />
4, Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional<br />
communication mediates a consensus in a microbial biofilm consortium. Proc<br />
Natl Acad Sci USA 104: 17300–17304.<br><br />
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<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-28T00:10:16Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
</th><br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to activate chloramphenicol resistance gene. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. In cell A (Fig 3-0-2), production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B (Fig3-0-3), production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. On the other hand, 3OC12HSL activates resistance gene of cell B and 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
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<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
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<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062, both of which are existing parts. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-28T00:08:35Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to activate chloramphenicol resistance gene. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. In cell A (Fig 3-0-2), production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B (Fig3-0-3), production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. On the other hand, 3OC12HSL activates resistance gene of cell B and 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
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<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062, both of which are existing parts. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance gene activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T23:56:29Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
<tr><br />
<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
</th><br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to activate chloramphenicol resistance gene. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. In cell A (Fig 3-0-2), production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B (Fig3-0-3), production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. On the other hand, 3OC12HSL activates resistance gene of cell B and 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
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<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
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<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062, both of which are existing parts. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T23:54:07Z<p>Kitano: </p>
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<font size="5" color="#eb8300"><b><center>Project menu</center></b></font><br />
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<table id="table-01"><br />
<tr><br />
<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to activate chloramphenicol resistance gene. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. In cell A (Fig 3-0-2), production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B (Fig3-0-3), production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. On the other hand, 3OC12HSL activates resistance gene of cell B and 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
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<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062, both of which are existing parts. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T23:49:23Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to activate chloramphenicol resistance gene. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. In cell A (Fig 3-0-2), production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B (Fig3-0-3), production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. On the other hand, 3OC12HSL activates resistance gene of cell B and 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062, both of which are existing parts. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T23:47:58Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to activate chloramphenicol resistance gene. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. In cell A (Fig 3-0-2), production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B (Fig3-0-3), production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. On the other hand, 3OC12HSL activates resistance gene of cell B and 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
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<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062, both of which are existing parts. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T23:44:59Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to activate chloramphenicol resistance gene. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. In cell A (Fig 3-0-2), production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B (Fig3-0-3), production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. On the other hand, 3OC12HSL activates resistance gene of cell B and 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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<FONT SIZE="6">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
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<FONT SIZE="6"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
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<FONT SIZE="6"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062, both of which are existing parts. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T23:10:55Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
We built Artificial Cooperation System to engineer E.coli with humanity. In this system quorum sensing system and chloramphenicol resistance protein 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 protein generator which is regulated by LuxR activation promoter. We confirmed that the cell with the chloramphenicol resistance protein generator can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed that LuxI can produce sufficient 3OC6HSL to help dying cell. Forth, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T22:57:45Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<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. 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 can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
<br />
== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T22:57:05Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<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. 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 can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T22:55:06Z<p>Kitano: /* luxI */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<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. 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 can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T22:44:51Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<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. 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 can survive under high concentration of chloramphenicol when 3OC6HSL exists. Third, we confirmed the feasibility of Artificial Cooperation System from simulation and experimental data.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and chloramphenicol resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and kanamycin resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, the expression of ''luxI'' is repressed by LasR/3OC12HSL complex. Therefore, the decrease of 3OC12HSL/LasR complex causes the overexpression of ''luxI'' and the increase of 3OC6HSL. This results in the expression of chloramphenicol resistance gene in the Cell A. Consequently, the Cell A is recovered and begins to produce the apple reporters in return!<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which play important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-1 shows that under the regulations by R0062 (LuxR activation promoter), gfp expression was activated in the presence of 3OC6HSL. Fig3-1-3 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig 3-2-1 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==luxI==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized existing LuxR repression promoter and LuxR activation promoter in the registry. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T22:10:16Z<p>Kitano: /* Conclusion */</p>
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
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.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and that of Cm resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and that of Kan resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which plays important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-2 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Fig3-1-1 shows that under the regulations by R0062,a LuxR activation promoter, gfp expression was activated in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig〇〇 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==luxI==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized R0061, R0062 and I14032. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity. <br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T21:36:31Z<p>Kitano: /* Abstract */</p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of cell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
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[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
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[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
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==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
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[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
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In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
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'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
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[[IMAGE:Tokyotech S9.jpg|400px]],<br />
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where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
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When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
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[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
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The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
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The growth inhibition by antibiotics is represented by the following equations:<br />
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[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
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where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
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The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
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[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
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Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
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[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
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'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
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[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
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Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
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[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
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where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
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'''III, AHL synthesis and degradation'''<br />
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AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
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[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
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where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
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'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
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[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
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Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
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[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
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Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
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==Prameters==<br />
The parameters used in equation S1 to S8 are listed in Table1. Many parameter values are taken from references. However, quantitative information is not sufficient for some parameters; therefore we use educated guesses that are biologically feasible. <br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
1, Bo Hu, Jin Du, Rui-yang Zou, Ying-jin Yuan, An Environment-Sensitive Synthetic Microbial Ecosystem, PLoS ONE, volume 5 issue 5, 2010<br><br />
2, Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187.<br><br />
3, Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.<br><br />
4, Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional<br />
communication mediates a consensus in a microbial biofilm consortium. Proc<br />
Natl Acad Sci USA 104: 17300–17304.<br><br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T21:08:35Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
</td><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
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.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and that of Cm resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and that of Kan resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which plays important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-2 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Fig3-1-1 shows that under the regulations by R0062,a LuxR activation promoter, gfp expression was activated in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig〇〇 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==luxI==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized R0061, R0062 and I14032. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity. And we noticed that we can make more interesting E. coli.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T21:07:45Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
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.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and that of Cm resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and that of Kan resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
<br />
== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which plays important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-2 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Fig3-1-1 shows that under the regulations by R0062,a LuxR activation promoter, gfp expression was activated in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig〇〇 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==luxI==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized R0061, R0062 and I14032. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity. And we noticed that we can make more interesting E. coli.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T21:05:31Z<p>Kitano: /* plux-CmR */</p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
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</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
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.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and that of Cm resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and that of Kan resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which plays important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-2 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Fig3-1-1 shows that under the regulations by R0062,a LuxR activation promoter, gfp expression was activated in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==Chloramphenicol resistance geneator activated by LuxR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig〇〇 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==luxI==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized R0061, R0062 and I14032. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity. And we noticed that we can make more interesting E. coli.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T20:43:00Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
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.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and that of Cm resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and that of Kan resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
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== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which plays important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-2 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Fig3-1-1 shows that under the regulations by R0062,a LuxR activation promoter, gfp expression was activated in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==plux-CmR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig〇〇 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==luxI==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized R0061, R0062 and I14032. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity. And we noticed that we can make more interesting E. coli.<br />
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<!-- --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_SystemTeam:Tokyo Tech/Project/Artificial Cooperation System2010-10-27T20:42:06Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>3 Artificial Cooperation System&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp;-YOU ARE HERE!-<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3 Artificial Cooperation System</b></font><br />
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=Artificial Cooperation System=<br />
=Abstract=<br />
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.<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
[[IMAGE:Tokyotech Fig.K395146-1.jpg|300px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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=Introduction=<br />
==What’s Artificial Cooperation System?==<br />
Artificial Cooperation System was built to engineer ''E. coli'' that behaves kindly and have sympathy. This system consists of two types 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. This is a summary of the Artificial Cooperation System.<br />
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[[Image:Tokyotech_top10.jpg|center|640px|thumb|Fig 3-0-1.They are competitors. 2.One is dying, another cell notices it. 3.The healthy cell rescues the dying cell. 4. The dying cell recovers and she gives "Apples" as expression of appreciation]]<br />
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==Genetic Circuit==<br />
===I, genetic circuit overview===<br />
We designed the following circuit. In Artificial Cooperation System, there are two types of ''E. coli'', Cell A and Cell B. Cell A has resistance to kanamycin and Cell B has resistance to chloramphenicol originally. <br />
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]<br />
[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]<br />
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Pcon: constitutive promoter.<br><br />
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.<br><br />
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.<br><br />
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br><br />
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br><br />
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br><br />
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br><br />
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and that of Cm resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and that of Kan resistance gene is activated by LasR/3OC12HSL complex. This means that 3OC12HSL produced by cell A and 3OC6HSL produced by cell B indirectly repress each other’s production. And 3OC12HSL activates resistance gene of cell B while 3OC6HSL activates resistance gene of cell A. Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is rescued and recovered.<br />
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<FONT SIZE="4">Normal</FONT><br />
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]<br />
[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]<br />
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In normal situation, Cell A and Cell B are competitors and recognize each other by using quorum sensing. First, LasI protein in Cell A produces 3OC12HSL. Second, these signal molecules diffuse through cell membranes and bind to LasR protein in Cell B. Third, LasR/3OC12HSL complex represses the production of LuxI protein. Similarly in cell B, LuxI protein produces 3OC6HSL. These signal molecules diffuse through cell membranes and bind to LuxR protein in Cell A. LuxR/3OC6HSL complex represses the production of LasI protein. Although Cell A and Cell B have chloramphenicol and kanamycin resistance gene respectively, they don’t express in this situation. This is because the concentration of 3OC12HSL and 3OC6HSL is too low to activate the expressions of kanamycin resistance gene and chloramphenicol resistance gene, respectively.<br><br />
<FONT SIZE="4"> when Cell A is dying </FONT><br />
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]<br />
[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br><br />
<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT><br />
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]<br />
[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]<br />
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]<br />
[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]<br />
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As we mentioned, originally Cell A has a resistance gene against kanamycin and does not have that against chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, the addition of chloramphenicol only decreases the number of Cell A. It means that 3OC12HSL decreases. It also results in the decrease of LasR/3OC12HSL complex that repress the expression of LasI in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.<br />
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=Result=<br />
In order to make the Artificial Cooperation System, we constructed and characterized several important parts and devices. First we characterized LuxR repression promoter R0061 and activation promoter R0062. Second, we designed and characterized a new LuxR repression promoter [http://partsregistry.org/Part:BBa_K395008 K395008]. Third, we designed and characterized a NEW BioBrick device [http://partsregistry.org/Part:BBa_K395162 K395162], a chloramphenicol resistance generator, which is activated by a LuxR activation promoter. Forth, we confirmed that LuxI can produce sufficient AHL to help dying cell. Finally, we simulated our Artificial Cooperation System and confirmed the feasibility of our system.<br />
<br />
== LuxR repression promoter and activation promoter==<br />
We characterized LuxR repression promoters and activation promoters, which plays important roles in the Artificial Cooperation System. The following figures are the result of experiments. Fig3-1-2 shows that under the regulations by R0061 and K395008, gfp expressions were repressed in the presence of 3OC6HSL. Fig3-1-1 shows that under the regulations by R0062,a LuxR activation promoter, gfp expression was activated in the presence of 3OC6HSL. Also, for realization of the Artificial Cooperative System work, the strength and the threshold of these promoters are so important. Thus we tried tuning promoters by changing sequence of consensus -35/-10 and lux box. [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|(See more…)]]<br />
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[[IMAGE:Tokyotech_plux_act_final.jpg|300px|left|thumb|fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)]]<br />
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[[IMAGE:Tokyotech_plux_rep_final.jpg|300px|right|thumb|fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)]]<br />
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==plux-CmR==<br />
We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator <br />
([http://partsregistry.org/Part:BBa_K395162 BBa_K395162]) which is activated by LuxR activation promoter. Fig〇〇 shows that in the presence of 3OC6HSL, the device is activated and as a result the cell was able to survive under high chloramphenicol concentration (750 ug/ml). [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|(See more…)]]<br />
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta]]<br />
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==luxI==<br />
We constructed a new LuxI generator ([http://partsregistry.org/Part:BBa_K395146 BBa_K395146]) and characterized its function. By combining I14032 (PlacI<sup>q</sup>) and K092400 (rbs-luxI-ter), we constructed K395146. Then we confirmed that the cell with K395146 produced the sufficient amount of 3OC6HSL to activate LuxR activation promoter (R0062) [[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|(See more…)]]<br />
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[[IMAGE:Tokyotech Fig.K395146-1.jpg|400px|left|thumb|Fig4 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano]]<br />
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==Mathematical modeling==<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 (Fig.3-0-2). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-0-3). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-0-4), 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 (Fig.3-0-5). From the above results, the feasibility of the Artificial Cooperation System was confirmed.[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|(See more…)]]<br />
[[IMAGE:tokyotech_no_anti_sim_result_cell.jpg|300px|left|thumb|Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano]] <br />
[[IMAGE:Tokyptech no system sim result.jpg|300px|left|thumb|Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano]] <br />
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[[IMAGE:tokyotech_cm_sim_result_cell.jpg|300px|left|thumb|Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano]]<br />
[[IMAGE:tokyotech_kan_sim_result_cell.jpg|300px|left|thumb|Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano]]<br />
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<!--このしたから--><br />
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=Conclusion=<br />
We succeeded in constructing two new BioBrick, LuxR repression promoter (K395008) and chloramphenicol resistance gene generator activated by LuxR and 3OC6HSL. We characterized R0061, R0062 and I14032. From the experimental data and simulation result, we confirmed the feasibility of the Artificial Cooperation System, E. coli with humanity. And we noticed that we can make more interesting E. coli.</div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T20:40:17Z<p>Kitano: /* Prameters */</p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<tr><br />
<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<div id="tf_SubWrapper"><br />
<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
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[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
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[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
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==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
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[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
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In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
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'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
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[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
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When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
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[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
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The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
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The growth inhibition by antibiotics is represented by the following equations:<br />
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[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
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where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
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The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
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[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
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Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
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[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
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'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
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[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
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Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
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[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
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where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
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'''III, AHL synthesis and degradation'''<br />
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AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
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[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
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where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
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'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
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[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
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Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
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<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
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Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
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==Prameters==<br />
The parameters used in equation S1 to S8 are listed in Table1. Many parameter values are taken from references. However, quantitative information is not sufficient for some parameters; therefore we use educated guesses that are biologically feasible. <br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
1, Bo Hu, Jin Du, Rui-yang Zou, Ying-jin Yuan, An Environment-Sensitive Synthetic Microbial Ecosystem, PLoS ONE, volume 5 issue 5, 2010<br><br />
2, Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187.<br><br />
3, Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.<br><br />
4, Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional<br />
communication mediates a consensus in a microbial biofilm consortium. Proc<br />
Natl Acad Sci USA 104: 17300–17304.<br><br />
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<!--ここまで書いて良いですよ--><br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T20:36:58Z<p>Kitano: /* References */</p>
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<tr><br />
<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
<tr><br />
<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
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[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
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==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
<br />
[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
<br />
In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
<br />
<br />
'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
<br />
<br />
[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
<br />
When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
<br />
[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
<br />
The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
<br />
The growth inhibition by antibiotics is represented by the following equations:<br />
<br />
[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
<br />
where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
<br />
<br />
The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
<br />
[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
<br />
Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
<br />
[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
<br />
<br />
<br />
'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
<br />
[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
<br />
Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
<br />
<br />
<br />
[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
<br />
where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
<br />
<br />
<br />
'''III, AHL synthesis and degradation'''<br />
<br />
<br />
AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
<br />
[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
<br />
where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
<br />
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'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
<br />
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[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
<br />
Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
<br />
<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
<br />
Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
<br />
==Prameters==<br />
Parameters we used to simulate the system is listed in Table1.<br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
1, Bo Hu, Jin Du, Rui-yang Zou, Ying-jin Yuan, An Environment-Sensitive Synthetic Microbial Ecosystem, PLoS ONE, volume 5 issue 5, 2010<br><br />
2, Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187.<br><br />
3, Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.<br><br />
4, Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional<br />
communication mediates a consensus in a microbial biofilm consortium. Proc<br />
Natl Acad Sci USA 104: 17300–17304.<br><br />
<br />
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<!--ここまで書いて良いですよ--><br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T20:36:17Z<p>Kitano: /* References */</p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
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[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
<br />
<br />
<br />
<br />
==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
<br />
[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
<br />
In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
<br />
<br />
'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
<br />
<br />
[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
<br />
When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
<br />
[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
<br />
The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
<br />
The growth inhibition by antibiotics is represented by the following equations:<br />
<br />
[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
<br />
where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
<br />
<br />
The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
<br />
[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
<br />
Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
<br />
[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
<br />
<br />
<br />
'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
<br />
[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
<br />
Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
<br />
<br />
<br />
[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
<br />
where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
<br />
<br />
<br />
'''III, AHL synthesis and degradation'''<br />
<br />
<br />
AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
<br />
[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
<br />
where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
<br />
<br />
<br />
'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
<br />
<br />
[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
<br />
Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
<br />
<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
<br />
Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
<br />
==Prameters==<br />
Parameters we used to simulate the system is listed in Table1.<br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
1, Bo Hu, Jin Du, Rui-yang Zou, Ying-jin Yuan, An Environment-Sensitive Synthetic Microbial Ecosystem, PLoS ONE, volume 5 issue 5, 2010<br><br />
2, Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187.<br><br />
3, Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.<br?<br />
4, Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional<br />
communication mediates a consensus in a microbial biofilm consortium. Proc<br />
Natl Acad Sci USA 104: 17300–17304.<br><br />
<br />
<br />
<br />
<br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T20:35:40Z<p>Kitano: /* References */</p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
<br />
<br />
<br />
<br />
==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
<br />
[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
<br />
In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
<br />
<br />
'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
<br />
<br />
[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
<br />
When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
<br />
[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
<br />
The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
<br />
The growth inhibition by antibiotics is represented by the following equations:<br />
<br />
[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
<br />
where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
<br />
<br />
The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
<br />
[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
<br />
Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
<br />
[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
<br />
<br />
<br />
'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
<br />
[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
<br />
Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
<br />
<br />
<br />
[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
<br />
where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
<br />
<br />
<br />
'''III, AHL synthesis and degradation'''<br />
<br />
<br />
AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
<br />
[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
<br />
where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
<br />
<br />
<br />
'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
<br />
<br />
[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
<br />
Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
<br />
<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
<br />
Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
<br />
==Prameters==<br />
Parameters we used to simulate the system is listed in Table1.<br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
1, Bo Hu, Jin Du, Rui-yang Zou, Ying-jin Yuan, An Environment-Sensitive Synthetic Microbial Ecosystem, PLoS ONE, volume 5 issue 5, 2010<br><br />
2, Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187.<br />
3, Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.<br />
4, Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional<br />
communication mediates a consensus in a microbial biofilm consortium. Proc<br />
Natl Acad Sci USA 104: 17300–17304.<br />
<br />
<br />
<br />
<br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T20:14:27Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
<tr><br />
<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
</tr><br />
<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<div id="tf_SubWrapper"><br />
<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
<br />
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<br />
<br />
<br />
<br />
<br />
[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
<br />
<br />
<br />
<br />
==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
<br />
[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
<br />
In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
<br />
<br />
'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
<br />
<br />
[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
<br />
When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
<br />
[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
<br />
The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
<br />
The growth inhibition by antibiotics is represented by the following equations:<br />
<br />
[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
<br />
where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
<br />
<br />
The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
<br />
[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
<br />
Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
<br />
[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
<br />
<br />
<br />
'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
<br />
[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
<br />
Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
<br />
<br />
<br />
[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
<br />
where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
<br />
<br />
<br />
'''III, AHL synthesis and degradation'''<br />
<br />
<br />
AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
<br />
[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
<br />
where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
<br />
<br />
<br />
'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
<br />
<br />
[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
<br />
Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
<br />
<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
<br />
Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
<br />
==Prameters==<br />
Parameters we used to simulate the system is listed in Table1.<br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T20:12:32Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
</td><br />
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<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
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<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
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<br />
<br />
<br />
<br />
[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
<br />
==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
<br />
[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
<br />
In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
<br />
<br />
'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
<br />
<br />
[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
<br />
When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
<br />
[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
<br />
The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
<br />
The growth inhibition by antibiotics is represented by the following equations:<br />
<br />
[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
<br />
where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
<br />
<br />
The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
<br />
[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
<br />
Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
<br />
[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
<br />
<br />
<br />
'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
<br />
[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
<br />
Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
<br />
<br />
<br />
[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
<br />
where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
<br />
<br />
<br />
'''III, AHL synthesis and degradation'''<br />
<br />
<br />
AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
<br />
[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
<br />
where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
<br />
<br />
<br />
'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
<br />
<br />
[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
<br />
Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
<br />
<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
<br />
Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
<br />
==Prameters==<br />
Parameters we used to simulate the system is listed in Table1.<br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T20:12:05Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
<tr><br />
<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
</td><br />
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</table><br />
</center><br />
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<div id="tf_SubWrapper"><br />
<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
<br />
==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
<br />
[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
<br />
In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
<br />
<br />
'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
<br />
<br />
[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
<br />
When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
<br />
[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
<br />
The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
<br />
The growth inhibition by antibiotics is represented by the following equations:<br />
<br />
[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
<br />
where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
<br />
<br />
The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
<br />
[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
<br />
Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
<br />
[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
<br />
<br />
<br />
'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
<br />
[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
<br />
Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
<br />
<br />
<br />
[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
<br />
where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
<br />
<br />
<br />
'''III, AHL synthesis and degradation'''<br />
<br />
<br />
AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
<br />
[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
<br />
where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
<br />
<br />
<br />
'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
<br />
<br />
[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
<br />
Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
<br />
<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
<br />
Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
<br />
==Prameters==<br />
Parameters we used to simulate the system is listed in Table1.<br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
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==References==<br />
<!--ここまで書いて良いですよ--><br />
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</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T20:10:31Z<p>Kitano: </p>
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<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
<tr><br />
<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
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<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
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<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
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[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
<br />
==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
<br />
[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
<br />
In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
<br />
<br />
'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
<br />
<br />
[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
<br />
When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
<br />
[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
<br />
The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
<br />
The growth inhibition by antibiotics is represented by the following equations:<br />
<br />
[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
<br />
where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
<br />
<br />
The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
<br />
[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
<br />
Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
<br />
[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
<br />
<br />
<br />
'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
<br />
[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
<br />
Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
<br />
<br />
<br />
[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
<br />
where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
<br />
<br />
<br />
'''III, AHL synthesis and degradation'''<br />
<br />
<br />
AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
<br />
[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
<br />
where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
<br />
<br />
<br />
'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
<br />
<br />
[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
<br />
Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
<br />
<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
<br />
Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
<br />
==Prameters==<br />
Parameters we used to simulate the system is listed in Table1.<br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
<br />
==References==<br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitanohttp://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modelingTeam:Tokyo Tech/Project/Artificial Cooperation System/modeling2010-10-27T20:09:32Z<p>Kitano: /* Abstract */</p>
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<tr><br />
<td>[[Team:Tokyo_Tech|1 Graphical abstract]]<br><br />
</td><br />
</tr><br />
<td>2 Apple reporter<br><br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]<br />
:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]<br />
</td><br />
<tr><br />
<th>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<br><br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 lux activation/repression promoter]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]<br />
:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]<br />
:3-4 modeling -YOU ARE HERE!-<br />
</th><br />
</tr><br />
<tr><br />
<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 wolfcoli overview]]<br><br />
:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 the new seriesof P''ompC'']]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]<br />
:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 wolfcoli system]]<br />
</td><br />
</tr><br />
</table><br />
</center><br />
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</div> <!-- tf_menu --><br />
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<div id="tf_SubWrapper"><br />
<font size="5"><b>3-4 modeling</b></font><br />
=Mathematical modeling=<br />
==Abstract==<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 (Fig.3-4-1). Second, when the system did not work and chloramphenicol existed, the number of sell A decreased while the number of cell B increased (Fig.3-4-2). Third, when the system worked and chloramphenicol existed, the number of cell A increased after temporal decline (Fig.3-4-3), 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 (Fig.3-4-4). From the above results, the feasibility of the Artificial Cooperation System was confirmed. <br />
<br />
[[IMAGE: tokyotech_no_anti_sim_result.jpg|300px|left|thumb|Fig3-4-1. When the system worked and any antibiotics did not exist (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_no_system_sim_result.jpg|300px|left|thumb|Fig3-4-2. when the system did not work and chloramphenicol existed (work done by Shohei Kitano)]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[IMAGE: tokyotech_cm_sim_result.jpg|300px|left|thumb|Fig3-4-3 when the system worked and chloramphenicol existed (work done by Shohei Kitano)]]<br />
[[IMAGE: tokyotech_kan_sim_result.jpg|300px|left|thumb|Fig3-4-4 when the system worked and Kanamycin existed (work done by Shohei Kitano) ]]<br />
<br />
==Model development==<br />
In order to simulate the Artificial Cooperation System, we developed an ordinary differential equation model. The state variables and parameters used in the model are described in detail in Table1. <br />
<br />
[[IMAGE:Tokyotech_S1-S8.jpg|600px]]<br />
<br />
In the Artificial Cooperation System, the major kinetic events are: cell population growth; resistance gene expression under the control of quorum sensing activation promoters; AHLs synthesis and degradation; I protein synthesis under the control of quorum sensing repressive promoters. These kinetic events are contained in the equations. Following sentences describe how the equations are developed.<br />
<br />
<br />
'''I, Cell Population Growth'''<br />
The growth rate of each type of cell without effect of antibiotics can be described by the following logistic growth equation:<br />
<br />
<br />
[[IMAGE:Tokyotech S9.jpg|400px]],<br />
<br />
where N denotes the number of cells, Nm denotes the maximum allowed number of cells due to nutrient limitation, and μ is a constant coefficient. The maximum growth rate is proportional to the number of cells and the growth rate is inhibited by the number of cells due to nutrient limitation. <br />
<br />
When two types of cell coexist, the growth rates of the both type of cell are described in the following equations: <br />
<br />
[[IMAGE:Tokyotech S10S11.jpg|400px]]<br />
<br />
The maximum growth rate of each type of cell is proportional to the number of their type of cells and the growths are inhibited by the total number of the cells in the system (1-(N<sub>A</sub>+ N<sub>B</sub>)/ N<sub>m</sub>)<br />
<br />
The growth inhibition by antibiotics is represented by the following equations:<br />
<br />
[[IMAGE:Tokyotech S12S13.jpg|400px]]<br />
<br />
where Cm and Kan denote chloramphenicol and kanamycin concentration respectively. The rate of decreasing the number of cells is proportional to the product of multiplying antibiotics concentration and the number of cell. Here, &gamma is the rate constant. <br />
<br />
<br />
The antibiotic resistance enzyme releases the antibiotic growth inhibition dependent on the enzyme concentration. Then, S12 and S13 are replaced by the following equations:<br />
<br />
[[IMAGE:Tokyotech S14S15.jpg|400px]]<br />
<br />
Where CmR and KanR are the concentration of the chloramphenicol and kanamycin resistance enzyme, respectively, and &eta; is constant for specific gene. Here, if (Cm-&eta;<sub>Cm</sub>CmR) or (Kan-&eta;<sub>Kan</sub>KanR) is negative, these terms are set to zero during simulation. As a consequence, the dynamics of the number of cells are described by equation S1 and S2.<br />
<br />
[[IMAGE:Tokyotech S1S2.jpg|400px]]<br />
<br />
<br />
<br />
'''II, Resistance gene expression under the control of AHLs'''<br />
In our system, the expression level of resistance gene is regulated by R protein-AHL complex. R proteins, LuxR and LasR, are assumed to be synthesized sufficiently so that the concentration of the complex depends on only the AHL concentration. Thus the synthesis rates of resistance enzyme are described by Hill function dependent on the AHL concentration (equation S16 and S17). <br />
<br />
[[IMAGE:Tokyotech S16S17.jpg|400px]]<br />
<br />
Together with the leaky production of the resistance enzymes and the degradation of resistance enzymes, equation S3 and S4 are employed to simulate the expression of resistance genes.<br />
<br />
<br />
<br />
[[IMAGE:Tokyotech S3S4.jpg|400px]]<br />
<br />
where leakyCmR and leakyKanR denote the leaky production of chloramphenicol and kanamycin resistance enzyme respectively, k<sub>CmR</sub> and k<sub>KanR</sub> denote the maximum synthesis rate of each enzyme, respectively. n<sub>1</sub> and n<sub>2</sub> are Hill coefficient. m<sub>Cm</sub> and m<sub>KanR</sub> denote the AHL concentration required for producing half level of each enzyme, respectively.<br />
<br />
<br />
<br />
'''III, AHL synthesis and degradation'''<br />
<br />
<br />
AHL is enzymatically synthesized by I protein from some substrates. For the simplicity, we assumed the amount of substrates is sufficient so that the AHL synthesis rate are estimated to be proportional to the cognate I protein. The degradation rate of AHL is assumed to decay with first-order kinetic. The dynamical behaviors of AHLs concentration are described by the following equations (S5 and S7):<br />
<br />
[[IMAGE:Tokyotech S5S7.jpg|400px]]<br />
<br />
where, k<sub>3OC6HSL</sub>, k<sub>3OC12HSL</sub>, d<sub>3OC6HSL</sub> and d<sub>3OC12HSL</sub> denote constant coefficients. <br />
<br />
<br />
<br />
'''IV, I protein production under the control of AHL dependent promoter'''<br />
The synthesis of I proteins can be generated by Hill function according to the concentration of AHL like the expression of resistance gene. Though the resistance genes are activated by AHLs, the production of I proteins is repressed by AHLs. So, we employ the following Hill function (equation S18 and S19)<br />
<br />
<br />
[[IMAGE:Tokyotech S18S19.jpg|400px]]<br />
<br />
Where, k<sub>LuxI</sub> and k<sub>LasI</sub> denote maximum production rate of LuxI and LasI. When the concentration of AHL is 0nM. n<sub>3</sub> and n<sub>4</sub> are Hill coefficients. m<sub>LuxI</sub> and m<sub>LasI</sub>denote the AHL concentration required for producing half level of I proteins. Therefore dynamics equations of I proteins production are listed in equation S6 and S8.<br />
<br />
<!--S6 S8--><br />
[[IMAGE:Tokyotech S6S8.jpg|400px]]<br />
<br />
Where d<sub>LuxI</sub>and d<sub>LasI</sub> denote constant coefficient.<br />
<br />
==Prameters==<br />
Parameters we used to simulate the system is listed in Table1.<br />
[[IMAGE:tokyotech_parameter_table.jpg|550px|left|thumb|Table1 Variables and Parameters]]<br />
<br />
<br />
<!--ここまで書いて良いですよ--><br />
</div> <!-- end SubWrapper --><br />
<br />
<br />
<br />
</div> <!-- end Super_main_wrapper --></div>Kitano