Team:Tokyo Tech/Project/Artificial Cooperation System

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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br>
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<td>2 Apple reporter<br>
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:[[Team:Tokyo_Tech/Project/Apple_Reporter|2-1 Color]]
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:[[Team:Tokyo_Tech/Project/Apple_Reporter2|2-2 Fragrance]]
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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>
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep|3-1 ''lux'' activation/repression promoter]]
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/Cm_assay|3-2 resistance gene activation device]]
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/luxI_assay|3-3 ''lux''I Assay]]
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:[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System/modeling|3-4 modeling]]
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<td>[[Team:Tokyo_Tech/Project/wolf_coli|4 Wolf coli overview]]<br>
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:[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|4-1 New seriesof P''ompC'']]
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:[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|4-2 lacIM1 for band-detect network ]]
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:[[Team:Tokyo_Tech/Project/wolf_coli/System|4-3 Wolf coli system]]
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==Artificial Cooperation System==
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<font size="5"><b>3 Artificial Cooperation System</b></font>
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=Requirement=
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In order to make our system, we need introduce cell-to-cell communication mechanism. Many cell-to-cell communication mechanisms in bacteria are known, though the mechanisms of many systems are not well-understood yet. Thus we used “quorum sensing” whose mechanism is well studied. And quorum sensing is popular in synthetic biology and iGEM.
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=Artificial Cooperation System=
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=Abstract=
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=quorum sensing=
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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.
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Quorum Sensing (QS) refers to cell-to-cell communication systems that are used by many microorganisms to sense their local cell densities. This sensing mechanism is based on the production, secretion and detection of small signaling molecules, whose concentration correlates to the abundance of secreting microorganisms in the vicinity. When the signal concentration reaches a threshold, they know the ‘quorum’ is formed, and the communicating microorganisms undergo a coordinated change in their gene-expression profiles. As a result, they initiate complicated activities which would not have been occurred at smaller cell numbers. Secretion of virulence factors, initiation of biofilm formation, sporulation, competence, mating, root nodulation, bioluminescence, and production of secondary metabolites are the examples for the activities mentioned above.
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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)]]
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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)]]
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[[Image:tokyotech_Cm-survival.jpg|300px|left|thumb|Fig3-2-1  Chloramphenicol resistance device. This work done by Yusuke Kaneta]]
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[[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]]
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=Introduction=
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==What’s Artificial Cooperation System?==
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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”.
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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]]
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==Genetic Circuit==
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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.
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[[IMAGE:Tokyotech cellA circuit.png|400px|left|thumb|Fig3-0-2.Genetic circuit in cell A]]
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[[IMAGE:Tokyotech cellB corcuit.png|400px|left|thumb|Fig3-0-3.Genetic circuit in cell B ]]
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There are two regulatory genes involved in QS, I and R genes. The I gene produces I protein and directs the synthesis of an N-acyl homoserine lactone (AHL). AHL is bacteria-specific and gene-specific signal molecule. On the other hand, R protein, which is expressed by R gene, binds to the synthesized AHL signal molecule. The AHL and R protein complex acts as a transcription factor.
 
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In synthetic biology field or iGEM, lux, las, and rhl have been used frequently.''lux'', ''lasI'' and ''rhlI'' gene synthesize N-(3-oxohexanoyl) homoserine lactone(3OC6HSL),  3-oxododecanoyl- homoserine lactone (3OC12HSL) and butanoyl- homoserine lactone (C4HSL) ,respectively. LuxR, LasR and RhlR receptor have been synthesized and can bind to 3OC6HSL, 3OC12HSL and C4HSL, respectively.
 
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In QS, not only activation promoters but also repression promoters have been used. The fact that LuxR and TraR promoter can function as both activation and repression has already been published in a paper*. We have characterized luxR promoter in order to confirm this feature of LuxR protein.
 
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* Conversion of the Vibrio fischeri Transcriptional Activator, LuxR, to a Repressor
 
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=Genetic circuit=
 
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'''I, genetic circuit overview'''<br>
 
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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.
 
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[[IMAGE:Tokyotech cellA circuit.png|400px]] [[IMAGE:Tokyotech cellB corcuit.png|400px]]
 
Pcon: constitutive promoter.<br>
Pcon: constitutive promoter.<br>
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Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br>
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.<br>
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br>
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.<br>
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lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.<br>
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luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.<br>
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<FONT SIZE="4">Normal</FONT>
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[[IMAGE:tokyotech_normal.jpg|400px|left|thumb|Fig3-0-4. Normal condition]]
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[[IMAGE: Tokyotech ppt normal.jpg|200px|thumb|Fig3-0-5. Normal condition]]
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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>
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<FONT SIZE="4"> when Cell A is dying </FONT>
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px|left|thumb|Fig 3-0-6. Cell A is dying.]]
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[[IMAGE: Tokyotech ppt cellA is dying.jpg|200px|thumb|Fig 3-0-7. Cell A is dying.]]
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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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<FONT SIZE="4"> when Cell B tries to rescue Cell A </FONT>
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[[IMAGE:tokyotech_rescue.png|400px||left|thumb|Fig 3-0-8. Cell B is trying to rescue cell A]]
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[[IMAGE: Tokyotech ppt cellB try.jpg|200px|thumb|Fig 3-0-9. Cell B is trying to resucue cell A]]
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[[IMAGE:tokyotech_apple_vol2.jpg|400px|left|thumb|Fig 3-0-10. Cell A is rescued by cell B.]]
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[[IMAGE: Tokyotech ppt cellA is helped.jpg|200px|thumb|Fig 3-0-11. Cell A is rescued by cell B.]]
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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!
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=Result=
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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.
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== LuxR repression promoter and activation promoter==
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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…)]]
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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)]]
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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)]]
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==Chloramphenicol resistance geneator activated by LuxR==
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We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (''CmR'') generator
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([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…)]]
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[[Image:tokyotech_Cm-survival.jpg|400px|left|thumb|Fig3-2-1  Chloramphenicol resistance device. This work done by Yusuke Kaneta]]
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==LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter==
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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…)]]
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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]]
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In cell A, production of LasI is repressed by LuxR/3OC6HSL complex and Cm resistance gene is activated by LuxR/3OC6HSL complex. In cell B, production of LuxI is repressed by LasR/3OC12HSL complex and Kan resistance gene is activated by LasR/3OC12HSL complex. This means AHL of cell A and cell B repress each other indirectly. And AHL of cell A activates resistance gene of cell B, and AHL of cell B activates resistance gene of cell A.  Apple gene is composed of crtEBIYZW and MpAAT1. This expresses only when the cell is helped.
 
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*'''normal'''
 
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[[IMAGE:tokyotech_normal.jpg|400px]]
 
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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/OC6HSL 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. That’s because the concentration of 3OC12HSL and 3OC6HSL is too low to activate kanamycin resistance gene and chloramphenicol resistance gene, respectively.
 
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*'''when Cell A is dying'''
 
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[[IMAGE:tokyotech_cell A is dyng.jpg|400px]]
 
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As we mentioned, originally Cell A has resistance to kanamycin and doesn’t have resistance to chloramphenicol. On the other hand, Cell B has resistance to chloramphenicol and doesn’t have resistance to kanamycin. Therefore, number of  only Cell A decreases after addition of chloramphenicol. This means 3OC12HSL decreases. And it also results in decreasing of 3OC12HSL and LasR complex in Cell B. By this process, Cell B notices that Cell A is dying and tries to rescue it.
 
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*'''when Cell B tries to rescue Cell A'''
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==Mathematical modeling==
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In order to confirm the feasibility of the Artificial Cooperation System, we simulated the system under typical four experimental conditions. First, when the system worked and any antibiotics did not exist, cell A and cell B in the system showed no difference of growth (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…)]]
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[[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]]
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[[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]]
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[[IMAGE:tokyotech_rescue.png|400px]]
 
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[[IMAGE:tokyotech_apple_vol2.jpg|400px]]
 
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As we mentioned, the expression of ''luxI'' is repressed by LasR and 3OC12HSL complex. Therefore, the decrease of 3OC12HSL and LasR complex leads to overexpression of ''luxI'' and 3OC6HSL. It results in the activation of chloramphenicol resistance gene and this is followed by increase of the number of Cell A. Consequently, the mission of rescuing Cell A is completed!
 
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'''II, strength and threshold of promoters are so important'''<br>
 
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To make our Artificial Cooperative System work, strength and threshold of promoters regulated by AHL are so important. For example, if the threshold of the resistance gene promoter is very low, resistance gene is activated by low AHL level. This means we can’t make a dangerous situation. And if the threshold of promoter of I gene is so high, the production level of I protein stays at highest level all the time. This means two types of cell help each other all the time. Even if the threshold of promoters is suitable, our system doesn’t work unless the strength of promoter is suitable. For example, when the strength of resistance gene promoter is low, cells can’t express adequate resistance gene and die. And if the strength of I protein promoter is low, cell can’t produce adequate amount of AHL to help the other cell. 
 
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Therefore, we have to design promoters whose threshold and strength are suitable for the Artificial Cooperative System.
 
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'''III, tuning the threshold and strength of promoter'''<br>
 
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To initiate transcription from Plux, LuxR needs to bind the regulatory region upstream of -35, lux box. In the absence of AHL, LuxR can’t bind lux box and can’t initiate transcription from Plux. However in the presence of AHL, AHL binds LuxR and then, LuxR/AHL complex can bind lux box and initiate transcription from Plux.
 
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Thus, how efficiently transcription initiate from Plux is determined by not only the concentration of LuxR/AHL, but the concentration of LuxR/AHL binding lux box. This concentration is determined by not only AHL, LuxR and LuxR/AHL concentration but also the binding affinity of LuxR/AHL complex and lux box. If the affinity is low, high concentration of LuxR/AHL complex is needed to initiate transcription efficiently. But if the affinity is high, high concentration of LuxR/AHL complex is not needed to initiate transcription efficiently. Therefore we can tune the AHL threshold of Plux by changing the sequence of lux box. These are represented by the following equations. And we can tune the strength of promoters by changing the sequence of -35 and -10 consensus sequence. Therefore we can obtain our ideal promoters by changing sequence.
 
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[[IMAGE:Tokyotech_plux_act_plux_rep.png|300px]]
 
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In these equations,βrepresents Plux max activity, complex represents the concentration of LuxR/3OC6HSL complex and Kd represents the affinity of LuxR and lux box. S1 and S2 represents that Kd doesn’t determine the Plux max activity, but determines the complex concentration producing half level from this promoter. So, changing sequence of lux box doesn’t lead changing the Plux max activity. This leads changing threshold of Plux. 
 
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β represents Plux max activity, thus β represents the strength of -35 and -10 sequence. Therefore changing sequence of -35 and -10 leads changing Plux max activity.
 
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=Works=
 
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'''I, characterization of R0061 (promoter repressed by LuxR/3OC6HSL)'''<br>
 
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First, we confirmed R0061, which is a existing BioBrick promoter repressed by LuxR/3OC6HSL complex. To confirm this promoter, we constructed following two plasmids (fig〇〇and fig〇〇)
 
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[[IMAGE:Tokyotech K395101.png|400px]]
 
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We introduced these two plasmid into DH5&alpha;. This ‘‘E. coli’’ expresses LuxR constitutively and has GFP under Plux rep, thus it is supposed that GFP expression is repressed when AHL exists. Fig〇〇 shows that R0061 works as we expected. [[
 
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[[IMAGE:tokyotech_LuxR repression promoter assay(R0061).jpg|400px]]
 
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In the presence of 3OC6HSL(100nM), the fluorescence is lower than in the absence of 3OC6HSL. We used a fusion of placIQ (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control. We measured fluorescence by FCM 3 hour after addition of 100nM 3OC6HSL.
 
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'''II, designing the new promoters repressed by LuxR/3OC6HSL, K395008 and K395009.'''<br>
 
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Though R0061 is repressed by LuxR/3OC6HSL, the leaky expression is so high. That’s because -35 and -10 sequence of R0061 is the same as the -35 and -10 sequence of J23119 whose strength is the highest in BioBrick constitutive promoters.
 
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[[IMAGE:Tokyotech J23119&R0061.png|400px]]
 
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Then we designed new BioBrick parts, K395008 and K395009, whose -35 and -10 sequence is different from R0061.
 
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The -35 and -10 sequence of K395008 is the same as that of J23108. The -35 and -10 sequence is the same as that of J23115. J23108 and J23115 are BioBrick constitutive promoters.
 
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[[IMAGE:Tokyotech_R0061&K395008&K395009.png|400px]]
 
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Why we chose J23108 and J23115 is that these expression levels are middle and low in BioBrick constitutive promoters. 2nd reason is that 3’ end nucleotide of -35 sequence of J23108 and J23115 is ‘A’ and that 5’ end nucleotide of -10 sequence 0f J23108 and J23115 is ‘T’. -35 and -10 overlap lux box.
 
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[[IMAGE:Tokyotech_J23108&J23115.png|400px]]
 
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'''III, characterization of K395008 (LuxR activation promoter)'''
 
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We assayed K395008, which is repressed by LuxR and 3OC6HSL. And this promoter is designed by us. To assay this promoter, we made a fusion of K395008 to K121013 which was used to assay R0061 and R0062 too. And we used S03119 on pSB3K3.
 
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[[IMAGE:Tokyotech K395105.png|400px]]
 
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After construction of K395195, we introduced K395195 and S03119 into DH5&alpha;. And we measured the fluorescence by FCM. Fig 〇〇shows this result.
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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]]
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[[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]]
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[[IMAGE:tokyotech_LuxR repression promoter assay(R0061weak).jpg|400px]]
 
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'''IV, characterization of R0062 (promoter activated by LuxR/3OC6HSL)'''
 
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First, we assayed R0062 which is an existing BioBrick promoter activated by LuxR/3OC6HSL complex. To confirm this promoter, we constructed following two plasmids (fig〇〇and fig〇〇)
 
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[[IMAGE:Tokyotech K395100.png|400px]]
 
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we introduced two plasmid into DH5&alpha;. This ‘‘E. coli’’ expresses LuxR constitutively and has GFP under Plux act, thus it is supposed that GFP expression is activated when 3OC6HSL exists. Fig〇〇 shows that these BioBricks, K395100 and S03119 (pSB3K3) , worked correctly. In the absence of AHL, the fluorescence is low. In the contrast, the fluorescence is so high in the presence of 3OC6HSL(100nM), that’s 〇〇fold higher than in the absence of 3OC6HSL.
 
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[[IMAGE:tokyotech_LuxR activation promoter assay(R0062).jpg|400px]]
 
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The previous experiment shows that Plux is worked. This means that Plux is activated by LuxR and 3OC6HSL. Next, we research the 3OC6HSL concentration dependence of Plux activity. We measured the fluorescence intensity under different concentration of AHL (0nM, 1nM, 3nM, 5nM, 10nM, 30nM, 50nM, 100
 
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[[IMAGE:tokyotech_LuxR ractivatio promoter assay2.jpg|400px]]
 
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Fig 〇〇is the result of measurement. This shows that the activity of R0062 is dependent on 3OC6HSL and the AHL threshold concentration is about 〇〇nM. We measured the fluorescence by FCM 3 hours after 3OC6HSL induction.
 
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=Conclusion=
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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.
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Latest revision as of 03:59, 28 October 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

3 Artificial Cooperation System

Contents

Artificial Cooperation System

Abstract

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.

fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)
fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)













Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta
Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano












Introduction

What’s Artificial Cooperation System?

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”.

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

Genetic Circuit

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.


Fig3-0-2.Genetic circuit in cell A
Fig3-0-3.Genetic circuit in cell B


















Pcon: constitutive promoter.
Plux act: promoter activated by LuxR and 3OC6HSL. We call this promoter luxR activation promoter.
Plux rep: promoter repressed by LuxR and 3OC6HSL. We call this promoter luxR repression promoter.
Plas act: promoter activated by LasR and 3OC12HSL. We call this promoter lasR activation promoter.
Plas rep: promoter repressed by LasR and 3OC12HSL. We call this promoter lasR repression promoter.
lasI: enzyme repressed by LuxR and 3OC6HSL. We call this promoter LuxR repression promoter.
luxI: enzyme repressed by LasR and 3OC12HSL. We call this promoter LasR repression promoter.



Normal

Fig3-0-4. Normal condition
Fig3-0-5. Normal condition















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.





when Cell A is dying

Fig 3-0-6. Cell A is dying.
Fig 3-0-7. Cell A is dying.














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.




when Cell B tries to rescue Cell A

Fig 3-0-8. Cell B is trying to rescue cell A
Fig 3-0-9. Cell B is trying to resucue cell A


Fig 3-0-10. Cell A is rescued by cell B.
Fig 3-0-11. Cell A is rescued by cell B.

























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!

Result

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 K395008. Third, we designed and characterized a NEW BioBrick device 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.

LuxR repression promoter and activation promoter

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. (See more…)

fig.3-1-1 luxR activation promoter assay (worked by Kitano Shohei & Eriko Uchikoshi)
fig.3-1-3 luxR repression promoter assay (worked by Shohei Kitano & Eriko Uchikoshi)






Chloramphenicol resistance geneator activated by LuxR

We succeeded in construction and characterization of a NEW Biobrick device of a chloramphenicol resistance (CmR) generator (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). (See more…)

Fig3-2-1 Chloramphenicol resistance device. This work done by Yusuke Kaneta

















LuxI can produce sufficient 3OC6HSL to activate LuxR activation promoter

We constructed a new LuxI generator (BBa_K395146) and characterized its function. By combining I14032 (PlacIq) 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) (See more…)

Fig3-3-1 LuxI and LuxR activation promoter assay. This work done by Eriko Uchikoshi and Shohei Kitano












Mathematical modeling

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.(See more…)

Fig3-0-2 Simulation result in absence of antibiotics. This work done by Shohei Kitano
Fig3-0-3 Simulation result without Artificial Cooperation System. This work done by Shohei Kitano













Fig3-0-4 Simulation result in the presence of chloramphenicol. This work done by Shohei Kitano
Fog3-0-5 Simulation result in the presence of kanamycin. This work done by Shohei Kitano












Conclusion

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.