Team:Tokyo Tech/Project/wolf coli

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[[Team:Tokyo_Tech/Project/wolf_coli/New_Series_of_PompC]]
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==Wolf coli==
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==Overview of new series of ''OmpC'' promoters==
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We constructed BioBrick parts of the new series of osmoregulative promoters which are derivatives of the wild type OmpC promoter. The new series of promoters are PompC(C), PompC(CB) and PompC(CS1). In order to measure the strength of each promoter we used GFP as a reporter. We found that expression of GFP in OmpC(CB) and OmpC(CS1) promoters increased in high osmolarity medium comparing with the expression in low osmolarity medium. In contrast, under same conditions, there is no significant difference of GFP expression in OmpC(C) and OmpC(WT) promoters.
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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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<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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<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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=New BioBrick Parts=
 
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[[Image:Van_OmpC_graph.jpg|left|thumb|400px|Figure 1. The induction of new'' OmpC'' series in high osmolarity medium ]]
 
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We succeeded in designing 2 news osmoregulative promoters, OmpC(CB) and OmpC(CS1), which can also be utilized in light sensitive system.
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<font size="5"><b>4 Wolf coli Overview</b></font>
==Introduction==
==Introduction==
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*'''What is ''OmpC'' promoter?'''
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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_OmpC_wild_type.jpg|center|400px|PompC(WT) and the two-component 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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''OmpC'' promoter, a part of our band detector circuit, plays a crucial role in initiating the transformation of sympathetic coli. into Werewolf coli. in light sensitive system. To accomplish band detector circuit in light sensitive system, varieties of OmpC promoters were designed and characterized in order to find an appropriate strength of the promoter particularly which can be activated by the “full moon light”.
 
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*'''The new series of ''OmpC'' promoters'''
 
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[[image:Tokyotech_New_pompC_construction.jpg||center|400px|PompC(WT) and the two-component system]]
 
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OmpC(WT) promoter (BBa_R0082) is regulated by phosphorylated ''OmpR'', a response regulator in EnvZ-OmpR 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.
 
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==Material and Method==
 
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;*Construction of E. coli strain MG1655
 
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Each new BioBrick part, PompC(C)-GFP, PompC(CB)-GFP and PompC(CS1)-GFP on pSB3K3 was introduced into E. coli strain MG1655. A strain containing pLacq-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.
 
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;*Medium
 
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Medium A, per liter, contained 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]
 
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;*Report assay
 
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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 OD590 reached 0.2, the fresh culture was diluted 1 : 3 into 2 ml of pre-warmed medium A. For high osmolarity conditions, the cultures were diluted with sucrose supplemented medium to the final concentration of 15% (wt/vol). [2]
 
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;*Sample preparation for GFP measurement
 
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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 OD590, 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 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 OD590.
 
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==Result==
 
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; Characterization of new series of ''OmpC'' propmoters
 
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[[Image:Van_OmpC_graph.jpg|thumb|left|400px|Figure 1. The induction of new'' OmpC'' series in high osmolarity medium ]]
 
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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 and PompC(WT)-GFP.
 
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==References==
 
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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>
 
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2. Batchelor E. & Goulian M. Imaging OmpR localization in Escherichia coli. Molecular Microbiology. 2006;59(6),1767-78 <br>
 
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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
 
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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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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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==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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==P''OmpC'' in red-light-dependent gene expression network==
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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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''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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[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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==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