Team:Tokyo Tech/Project/wolf coli

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<font size="5" color="#eb8300"><b><center>Project menu</center></b></font>
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[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC|PompC]]
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[[Team:Tokyo_Tech/Project/wolf_coli/lacIM1|lacIM1]]
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=Wolf coli=
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=Abstract=
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It came to our mind that changing the regulator of ACS, we could endow E.coli with complicated behavior.  As a first step of our challenge, we aimed to design “Wolfcoli” inspired by famous legend of wolfman.  When there is intermediate light intensity, two cells doesn’t help each other at only full-moon night .  From these behaviors, we call this “Wolfcoli” (Fig. 3-1).  In our attempt to produce a “wolfcoli”, we introduced the "a red-light-dependent gene expression network"[1] and "band-detect network"[2] into the Artificial Cooperation System (Fig. 3-2).  We conducted experiment about PompC and LacIM1, which are key parts of each network. 
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We succeeded in designing 2 news osmoregulative promoters, POmpC(CB) and POmpC(CS1), which can also be utilized in the red-light-dependent gene expression network (Fig. 3-3). (詳しくは下へ)
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We have characterized lacIM1 (BBa_K082026) which is the key component in the band-detect network.  Though this part was registered by USTC(2008) [3], it was not well characterized in their wiki.
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We found that lacIM1 shows weaker repression than WT (Fig. 3-4). (詳しくは下へ)
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[[Image:tokyotech_wolfcoli_system_ver2.png|left|thumb|200px|Figure3-1. Introduction of “Wolfcoli”]]
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[[Image:tokyotech_LacIM1_system_ver3.png|left|thumb|200px|Figure3-2. Overview of “Wolfcoli” system]]
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[[Image:tokyotech_ompC_data_ver2.png|left|thumb|200px|Figure3-3. The induction of new'' OmpC'' series in high osmolarity medium.  This work is done by Thiprampai Thamamongood ]]
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[[Image: tokyotech_LacIM1_data_ver2.png|left|thumb|200px|Figure3-4. Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG.  This work is done by Mitsuhiko Odera ]]
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<center>
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<table id="table-01">
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<td>[[Team:Tokyo_Tech|1 Graphic abstract]]<br>
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</td>
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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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</td>
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<tr>
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<td>[[Team:Tokyo_Tech/Project/Artificial_Cooperation_System|3 Artificial Cooperation System]]<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>
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<tr>
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<th>4 Wolf coli overview  -YOU ARE HERE!-<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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</table>
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</center>
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<font size="5"><b>4 Wolf coli Overview</b></font>
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==Introduction==
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[[Image:tokyotech_wolfcoli_system_ver2.png|left|thumb|250px|Fig. 4-1 Cooperative activity of Artificial Cooperation System]]
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[[Image:tokyotech_wolfcoli_system_ver5.png|right|thumb|350px|Figure 4-2. Overview of “Wolf coli” system]]
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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.
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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]]
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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]]
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=Introduction=
 
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==What is “Wolfcoli” ?==
 
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As I mentioned before, in normal situation, when the Artificial Cooperation System is on, two cells are able to communicate each other, and help the other cell. On the other hand, when the system is off, two cells are competitors and recognize each other by using quorum sensing. (Artificial Cooperation System 参照)
 
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In order to assemble a more intelligible and imaginable system, we linked our project to a well-known character ”Wolfman”.  When there is intermediate light intensity, two cells doesn’t help each other at only full-moon night .  From these behaviors, we call this “Wolfcoli” (Fig. 3-1).
 
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  [[Image:tokyotech_wolfcoli_system_ver2.png|center|thumb|350px|Figure3-1. Introduction of “Wolfcoli”]]
 
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・At crescent moon night (Weak light intensity)<br>
 
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When there is weak light intensity, Artificial Cooperation System turns on, and two cells are able to communicate.  Then, two types of cell help each other. <br>
 
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・At daytime  (Strong light intensity)<br>
 
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When there is strong light intensity, Artificial Cooperation System turns on, and two cells are able to communicate, too.  Then, two types of cell also help each other. <br>
 
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・At full moon night  (Intermediate light intensity)<br>
 
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When there is intermediate light intensity, Artificial Cooperation System turns off, and two cells can’t communicate. two cells start competing with each other at full-moon night .  From these behavior, we call this “Wolfcoli”.
 
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This system is able to be accomplished by integrating the red-light-dependent gene expression network(3階層目へ) and the band-detect network (3階層目へ) (Fig. 3-2).
 
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[[Image:tokyotech_LacIM1_system_ver3.png|center|thumb|350px|Figure3-2. Overview of “Wolfcoli” system]]
 
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(また、Wolfcoliの詳しい説明はこちらへ)
 
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=Results=
 
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We conducted experiment about PompC and LacIM1, which are key parts of each network.
 
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==1.Characterization of new series of PompC==
 
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We succeeded in designing 2 news osmoregulative promoters, P''OmpC(CB)'' and P''OmpC(CS1)'', which can also be utilized in the red-light-dependent gene expression network. (Fig. 3-3) (詳しくはこちらへ) <br>
 
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[[Image:tokyotech_ompC_data_ver2.png|center|thumb|350px|Figure3-3. The induction of new'' OmpC'' series in high osmolarity medium.  This work is done by Thiprampai Thamamongood ]]
 
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==2.Characterization of LacIM1(BBa_K082026)==
 
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We confirmed that lacIM1 shows weaker repression than WT (Fig. 3-4) (詳しくはこちらへ)
 
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<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)
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[[Image: tokyotech_LacIM1_data_ver2.png|center|thumb|350px|Figure3-4. Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG.  This work is done by Mitsuhiko Odera ]]
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==Wolf coli and Artificial Cooperative System ==
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[[Image:tokyotech_wolfcoli_system_ver2.png|thumb|center|350px|Fig. 4-1 Cooperative activity of Artificial Cooperation System]]
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The Artificial Cooperation System was designed to be switched off during the “full moon night”. Therefore, Sympathetic coli would  transform into Wolf coli at the “full moon light”. During this period, communication between 2 types of cells doesn't occur. 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 switches the Artificial Cooperation System on, thus two types of cells are able to communicate and help each other. “Medium light” during the full moon night can switch the system off resulting in appearance of the “Wolfcoli”. During the daytime, “strong light” from the sun activates the Artificial Cooperation System.
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=Conclussion=
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==P''OmpC'' in red-light-dependent gene expression network==
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<br>We characterized key parts of building the “Wolfcoli” system.
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[[Image:Tokyotech wolfcoli system_ver5.png|left|thumb|300px|Fig. 4-5 Overview of red-light-dependent gene expression network in Wolf coli system]]
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[[Image:Tokyotech_ompc_graph.jpg|thumb|left|320px|Fig. 4-6 The activation of new P''ompC'' series in high osmolarity medium at 4 hours. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA]]
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=Reference=
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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 Wolf coli. To accomplish band-detect circuit in light sensing 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)
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1. Levskaya A, Chevalier AA, Tabor JJ, et al. Engineering Escherichia coli to see light. NATURE  2005;438, 441-442 <br>
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[http://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC#Introduction ...see more about ''OmpC'' promoter ](Fig. 4-6)
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2. 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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3.USTC(2008)
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==LacIMI in band-detect network==
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[[image:Tokyotech_band_detect.png|340px|thumb|left| Fig.4-7 Patial circuit of band-detect network ]]
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[[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]]
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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). [http://2010.igem.org/Team:Tokyo_Tech/Project/wolf_coli/lacIM1 …see more about lacIM1].
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==References==
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[1]. Basu S, Gerchman Y, Collins CH, et al. A synthetic multicellular system for programmed pattern formation. NATURE 2005;434,1130-1134
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Latest revision as of 03:56, 28 October 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

4 Wolf coli Overview

Contents

Introduction

Fig. 4-1 Cooperative activity of Artificial Cooperation System
Figure 4-2. Overview of “Wolf coli” system












Have you heard the legend of 'The Wolfman'? They're ordinary man at daytime, but suddenly transform into a ferocious wolf in the full-moon night. Our project aim to imitate the character of Wolfman, more specifically, designing two types of E.coli that helps each other to survive at daytime, whereas competing at full moon night. In order to create the “Wolf coli”, we introduced " red-light-dependent gene expression network"[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.

Fig. 4-3 The induction of new PompC series in high osmolarity medium at 4 hours. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA
Fig. 4-4 Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG. This work is done by Mitsuhiko ODERA












We have succeeded in designing 2 new osmoregulative promoters, PompC(CB) (BBa_K395302) and PompC(CS1)(BBa_K395303), which can be utilized in the red-light-dependent gene expression network (Fig. 4-3). We also characterized LacIM1 (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)

Wolf coli and Artificial Cooperative System

Fig. 4-1 Cooperative activity of Artificial Cooperation System

The Artificial Cooperation System was designed to be switched off during the “full moon night”. Therefore, Sympathetic coli would transform into Wolf coli at the “full moon light”. During this period, communication between 2 types of cells doesn't occur. 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 switches the Artificial Cooperation System on, thus two types of cells are able to communicate and help each other. “Medium light” during the full moon night can switch the system off resulting in appearance of the “Wolfcoli”. During the daytime, “strong light” from the sun activates the Artificial Cooperation System.

POmpC in red-light-dependent gene expression network

Fig. 4-5 Overview of red-light-dependent gene expression network in Wolf coli system
Fig. 4-6 The activation of new PompC series in high osmolarity medium at 4 hours. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA


OmpC promoter, in red-light-dependent gene expression network, plays a crucial role in initiating the transformation of Sympathetic coli into Wolf coli. To accomplish band-detect circuit in light sensing 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) ...see more about OmpC promoter (Fig. 4-6)

LacIMI in band-detect network

Fig.4-7 Patial circuit of band-detect network
Fig. 4-8.Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG. This work is done by Mitsuhiko ODERA













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). …see more about lacIM1.

References

[1]. Basu S, Gerchman Y, Collins CH, et al. A synthetic multicellular system for programmed pattern formation. NATURE 2005;434,1130-1134