Team:Grenoble-EMSE-LSU/Project

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<h2>Principle</h2>
<h2>Principle</h2>
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<p>Our system is based on KillerRed (KR), a fluorescent protein that produces Reactive Oxygen Species (ROS) upon illumination with green light <a href="#ref_bio_ov">[1]</a>. ROS, such as hydrogen peroxide (H<sub>2</sub>0<sub>2</sub>) or singlet oxygen (1O<sub>2</sub>), react with bacterial DNA and proteins, causing irreversible damages that ultimately lead to cell death (figure 2). Using KillerRed with illumination at different light intensities, we aim to control variations in the number of living <em>E. coli</em> bacteria of a liquid culture (Fig 1.).<br><br></p>
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<p>Our system is based on KillerRed, a fluorescent protein that produces Reactive Oxygen Species (ROS) upon illumination with green light <a href="#ref_bio_ov">[1]</a>. ROS, such as hydrogen peroxide (H<sub>2</sub>0<sub>2</sub>) or singlet oxygen (1O<sub>2</sub>), react with bacterial DNA and proteins, causing irreversible damages that ultimately lead to cell death (Fig 2.). Using KillerRed with illumination at different light intensities, we aim to control variations in the number of living <em>E. coli</em> bacteria of a liquid culture (Fig 1.).<br><br></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/5/5d/Grenoble_courbe_drawing.png" alt="" width="600px"></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/5/5d/Grenoble_courbe_drawing.png" alt="" width="600px"></p>
                                         <p id="legend">Figure 1.<br>Light-mediated control of the living biomass. Increase, decrease or stabilization of the number of living cells occurs in response to light stimulations at a carefully selected intensity.<br><br></p>
                                         <p id="legend">Figure 1.<br>Light-mediated control of the living biomass. Increase, decrease or stabilization of the number of living cells occurs in response to light stimulations at a carefully selected intensity.<br><br></p>
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<p>In this project, the expression of the KillerRed gene is placed under control of the Cph8/OmpC/pOmpC red-sensitive gene expression system <a href="#ref_bio_ov">[2]</a>. Therefore, both KR production and ROS-mediated cell death can be triggered with appropriate light stimulations (Fig 2.).<br><br></p>
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<p>In this project, the expression of the KillerRed gene is placed under control of the Cph8/OmpC/pOmpC red-sensitive gene expression system <a href="#ref_bio_ov">[2]</a>. Therefore, both KillerRed production and ROS-mediated cell death can be triggered with appropriate light stimulations (Fig 2.).<br><br></p>
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                                         <p align="center"><img src="" alt="" height="350px"></p>
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                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/1/1b/Grenoble_Red_induced.png" alt="" height="350px"></p>
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                                         <p id="legend">Figure 2.<br>Overview on our genetic network (copy of the genetic network of an iGEM team from a previous edition of the competition. This sketch is quite simple and easy to understand. We could try doing something similar for presenting the whole network simply and clearly (Voigt’s system is complicated, and we probably don’t need to detail the complete set of genetic sequences involved in his sensors).<br><br></p>
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                                         <p id="legend">Figure 2.<br>Overview on our genetic network <a href="#ref_bio_ov">[2,3]</a><br><br></p>
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<p>This optogenetic system enables us to interface the cell culture with TalkE’coli, an electronic device that can send orders to bacteria (“produce KR”, “die”) via a light source, all while monitoring their fluorescence via a photodiode.<br>
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<p>In response to green/white light stimulation, the KillerRed protein produces ROS that damage endogenous DNA and proteins, leading to cell death. The expression of the KillerRed gene is controlled via the cph8/OmpR/pOmpC red light-sensitive transcription system [2]. pLTetO-1 and pLac/ara-1 allow for the expression of the transmembrane protein cph8 in its phosphorylated ground state and of the phycocyanobilin chromophore, respectively [2]. In its phosphorylated state, cph8 triggers phosphorylation of the regulatory protein OmpR, which activates transcription of the cI repressor gene. cI can be considered as a not gate that represses the expression of the KillerRed gene.<br><br>
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                                        Light stimulation at 650 nm enables dephosphorylation of cph8 and thus to bypass the repression system, ultimately leading to the expression of the KillerRed protein.<br><br>
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                                        The use of optogenetic tools such as KillerRed and the cph8/OmpR/pOmpC gene transcription system enable us to interface our biological system with an optoelectonic device, equipped with a light source, that can send orders to the system ("die" or "produce KillerRed") via a light source, all while monitoring their fluorescence via a photodiode.<br><br>
                                         In order to fine-tune the device, several initial biological experiments were performed in order to gather data that could be effectively modeled. Modeling these experiments allowed us to identify specific parameters that could be focused upon in further biological tests. This key interplay between biology and modeling pushed our project to the next level, allowing the team to improve our experiments at each phase and to strive for better and better results.</p>
                                         In order to fine-tune the device, several initial biological experiments were performed in order to gather data that could be effectively modeled. Modeling these experiments allowed us to identify specific parameters that could be focused upon in further biological tests. This key interplay between biology and modeling pushed our project to the next level, allowing the team to improve our experiments at each phase and to strive for better and better results.</p>
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<h2>Genetic Network</h2>
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                                         In summary, iGEM Grenoble-EMSE-LSU:
                                         In summary, iGEM Grenoble-EMSE-LSU:
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     <li>Built two new biobricks for the KR Module: pLac-RBS-KR and pLac-RBS-mCherry</li>
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     <li>Built two new biobricks for the KillerRed Module: pLac-RBS-KR and pLac-RBS-mCherry</li>
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     <li>Prooved the expression of KillerRed (KR) in <em>E. coli</em>, while demonstrating that the number of living cells could be controlled with light illumination at different intensities</li>
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     <li>Prooved the expression of KillerRed in <em>E. coli</em>, while demonstrating that the number of living cells could be controlled with light illumination at different intensities</li>
     <li>Developed a predictive model in order to derive the intensity function, which achieves the desired variation in the number of living cells</li>
     <li>Developed a predictive model in order to derive the intensity function, which achieves the desired variation in the number of living cells</li>
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    <li>Built the electronic interfacing device TalkE’coli, so that a user can maintain a desired concentration of living cells within a sample with our engineered bacteria. It is mountable in an incubator and allows you to stabilize the living cell density</li>
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    <li>Developed an interactive video game that aims to raise awareness about Synthetic Biology and the iGEM competition</li>
     <li>Performed additional qualitative characterization experiments based on the Voigt system</li>
     <li>Performed additional qualitative characterization experiments based on the Voigt system</li>
     <li>Built 3 new biobricks for the Voigt Module: Redsensor-mRFP, Redsensor-KR, Greensensor-GFP that may enable the dynamic and quantitative investigation of Voigt’s photosensitive systems</li>
     <li>Built 3 new biobricks for the Voigt Module: Redsensor-mRFP, Redsensor-KR, Greensensor-GFP that may enable the dynamic and quantitative investigation of Voigt’s photosensitive systems</li>
     <li>Built the biobrick pBad-sspB for the Degredation Module: This could be of interest in case of a high concentration of intracellular KR, which could prevent bacteria from growing in any light conditions</li>
     <li>Built the biobrick pBad-sspB for the Degredation Module: This could be of interest in case of a high concentration of intracellular KR, which could prevent bacteria from growing in any light conditions</li>
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    <li>Add Nicolas box</li>
 
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    <li>add the videogame</li>
 
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    <li>anything alse, sellable?!</li>
 
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                                         <h2 id="ref_bio_ov">References</h2>
                                         <h2 id="ref_bio_ov">References</h2>
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                                         <p><strong>[1]</strong> M.E. Bulina et al., A genetically encoded photosensitizer, <em>Nature Biotechnology</em>, January 2006<br>
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                                         <p><strong>[1]</strong> M.E. Bulina <em>et al.</em>, A genetically encoded photosensitizer, <em>Nature Biotechnology</em>, January 2006.<br>
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                                           <strong>[2]</strong> J.J. Tabor et al., Multichromatic Control of Gene Expression in Escherichia coli, <em>Journal of Molecular Biology</em>, 2011</p>
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                                           <strong>[2]</strong> J.J. Tabor <em>et al.</em>, Multichromatic Control of Gene Expression in Escherichia coli, <em>Journal of Molecular Biology</em>, 2011.<br>
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                                          <strong>[3]</strong> https://2011.igem.org/Team:TU_Munich/project/introduction</p>
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Latest revision as of 03:35, 5 October 2013

Grenoble-EMSE-LSU, iGEM


Grenoble-EMSE-LSU, iGEM

Retrieved from "http://2013.igem.org/Team:Grenoble-EMSE-LSU/Project"