Difference between revisions of "Team:NAWI Graz/Description"

 
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         <h1>PROJECT DESCRIPTION</h1>
 
         <h1>PROJECT DESCRIPTION</h1>
 
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             The aim of our project was to create a robot-bacteria interface, where information processing is done by a bacterial culture
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             The aim of project <command class="colibot"/> was to create a robot-bacteria interface. Information processing is done by a bacterial culture that exhibits a feedback loop with a mobile robot. To enable communication between bacteria and robot, we decided to engineer <i>Escherichia coli</i> cells to respond to environmental changes with an increase in fluorescence in the culture. To achieve environment-dependent expression of fluorescence proteins, we used the temperature-inducible ibpA-promoter and two promoters which respond to shifts in pH, the acid-inducible asr-promoter and the alkaline-inducible alx-promoter.  
            that exhibits a feedback loop with a mobile robot. To enable communication between bacteria and robot, we decided to engineer <i>E. coli</i> cells to respond to changes in pH with a change in fluorescence. To achieve pH-dependent expression of fluorescence protein, we used two different promoters: the acid-inducible <i>asr</i> promoter and the alkaline inducible <i>alx</i> promoter.
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            <source src="https://static.igem.org/mediawiki/2017/3/38/Wikidescr.mp4" type="video/mp4"> Your browser does not support the video tag.
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<b>Vid. 1:</b> Video about our <command class="colibot"/> project. This video gives a short overview of project <command class="colibot"/>.
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         <h2 class="section-sub">Introduction</h2>
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         <h2 class="section-sub">Project <command class="colibot"/></h2>
 
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            An important area at the cutting edge of technology is the integration of biological systems in mechanical and automated
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          An important area at the cutting edge of technology is the integration of biological systems in mechanical and automated systems. For our iGEM project we therefore chose to take a new approach on the integration of biology into technology. Our goal was the creation of a robot-bacteria interface that acts in a constant feedback loop. The first step was to make communication between bacteria and a technical system possible. Therefore, we engineered <i>E. coli</i> cells, which are sensitive to certain environmental conditions. When these conditions are met, the bacteria will express certain detectable proteins.  
            systems. For our iGEM project we therefore chose to take a new approach on the integration of biology into technology.
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In our setup a mobile robot moves through an arena and uses its proximity sensors to measure whether there is a wall in front of it. If the robot detects a wall, a signal will be transferred to a bacterial culture in the form of an environmental change. To provide them with stable conditions, the bacteria are cultivated in a <a href="https://2017.igem.org/Team:NAWI_Graz/Bioreactor">bioreactor</a>. In the bacteria, promoters sensitive to the these environmental changes will be activated and promote the expression of a fluorescent protein. The resulting fluorescence signal can be detected by an optical system and relayed back to the robot resulting in a response in form of a directional change.  
            Our goal is the creation of a bacteria computer interface that acts in a constant feedback loop. We create a
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For the realization of this concept we created a <a href="https://2017.igem.org/Team:NAWI_Graz/TemperaturePlasmid">temperature-sensitive construct</a> first. The heat shock promoter ibpA controls the expression of one fluorescent protein, which allows a simple yes/no decision. We used it for preliminary experiments to move a robot through a maze. To expand the possibilities of communication we created <a href="https://2017.igem.org/Team:NAWI_Graz/pHPlasmid">constructs sensitive to acidic and alkaline pH</a>. The acid-inducible asr-promoter and the alkaline-inducible alx-promoter are used to control the expression of two different fluorescent proteins. A shift in pH-value will lead to a change of fluorescence and allow navigation of the robot in a more sophisticed way.  
            pH sensitive E. coli strain that expresses detectable proteins according to a pH level. The pH level is determined
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            by the position of a mobile robot in an arena with a virtual pH gradient. The pH level of the robot's position
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            is passed on to bacteria that grow in a bioreactor, as a response to the pH shift, a shift in protein expression
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            is made due to pH sensitive promoters. This altered expression correlates with a change in color, due to a change
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            in expression, that is detectable with an optical system. The data from the optical signal is then send back
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            to the robot resulting in a response in form of a directional change. Thus, we can control the motion of the
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            robot through the arena with a constant feedback loop to the bioreactor. Furthermore, it is planned, to characterize
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            and develop one of the used pH sensitive promotors, as a Biobrick.
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        <h2 class="section-sub">Detailed Project Description</h2>
 
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            It is planned to design a pH-sensitive E. coli strain with two different plasmids. Each of the plasmids contains a pH sensitive
 
            promotor. The promotors are induced by different pH levels. An acidic pH leads to the expression of a red fluorecent
 
            protein and an alkaline pH to a green fluorecent protein. Both fluorecent proteins contain a N-terminal TEV-site
 
            followed by a F-degron. The TEV-site, which allows the degradation by the TEV-protease. The F-degron should increase
 
            the velocity of the degradation of the fluorecent protein by recruiting endogenous ClpAP machinery. This is important
 
            to provide a faster color change due to pH shift. The TEV-protease is integrated into the genome by CRISPR/Cas9.
 
            Due to the assay, it is necessary that the proteins are produced and degraded very fast. Therefor, the protein
 
            exhibits a quick maturation time. Based on the combination of the TEV-protease and a quick maturation time, it
 
            is possible to gain rather quick changes of the fluorescence in the media. The fluorescent signals are detected
 
            by a camera, leading to maneuvering the robot through an arena. Depending on the fluorescent color of the media,
 
            the robot turns either left or right. For a better performance of transcription and expression the acid-inducable
 
            asr-promotor should be characterized and further developed. Therefor, it is planned to mutate some regions, like
 
            the RBS (ribosome binding site) of the promotor to optimized it.
 
 
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        <h2 class="section-sub">Applications and Implications</h2>
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            One of the possible applications could be a bioreactor with bacteria that control their environment via active feedback.
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            Meaning that the bacterias give a signal, if they are in need for a different media composition, for example
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            if more nutrients or a switch of the pH level become necessary bacteria indicate it and a robot automatically
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            adapts the medium to the circumstances. The technology also holds potential in the creation of reusable biosensors
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            or a controlled bioremediation effort, where bacteria are mobile due to the connection to a robot host.
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Latest revision as of 01:51, 2 November 2017

PROJECT DESCRIPTION

The aim of project was to create a robot-bacteria interface. Information processing is done by a bacterial culture that exhibits a feedback loop with a mobile robot. To enable communication between bacteria and robot, we decided to engineer Escherichia coli cells to respond to environmental changes with an increase in fluorescence in the culture. To achieve environment-dependent expression of fluorescence proteins, we used the temperature-inducible ibpA-promoter and two promoters which respond to shifts in pH, the acid-inducible asr-promoter and the alkaline-inducible alx-promoter.

Vid. 1: Video about our project. This video gives a short overview of project .

Project

An important area at the cutting edge of technology is the integration of biological systems in mechanical and automated systems. For our iGEM project we therefore chose to take a new approach on the integration of biology into technology. Our goal was the creation of a robot-bacteria interface that acts in a constant feedback loop. The first step was to make communication between bacteria and a technical system possible. Therefore, we engineered E. coli cells, which are sensitive to certain environmental conditions. When these conditions are met, the bacteria will express certain detectable proteins. In our setup a mobile robot moves through an arena and uses its proximity sensors to measure whether there is a wall in front of it. If the robot detects a wall, a signal will be transferred to a bacterial culture in the form of an environmental change. To provide them with stable conditions, the bacteria are cultivated in a bioreactor. In the bacteria, promoters sensitive to the these environmental changes will be activated and promote the expression of a fluorescent protein. The resulting fluorescence signal can be detected by an optical system and relayed back to the robot resulting in a response in form of a directional change. For the realization of this concept we created a temperature-sensitive construct first. The heat shock promoter ibpA controls the expression of one fluorescent protein, which allows a simple yes/no decision. We used it for preliminary experiments to move a robot through a maze. To expand the possibilities of communication we created constructs sensitive to acidic and alkaline pH. The acid-inducible asr-promoter and the alkaline-inducible alx-promoter are used to control the expression of two different fluorescent proteins. A shift in pH-value will lead to a change of fluorescence and allow navigation of the robot in a more sophisticed way.