Team:Grenoble-EMSE-LSU/Project/Instrumentation

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<h1>TalkE'coli - Our device</h1>
<h1>TalkE'coli - Our device</h1>
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<strong>DO NOT EDIT THIS PAGE!!!</strong> </br>
 
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                                             <h2 id="Overview">Overview of the device</h2>
                                             <h2 id="Overview">Overview of the device</h2>
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      <p>Our project aims to <strong>control the concentration of living bacteria in a culture</strong>. To do so, we designed <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology">a genetic network allowing light controlled cell growth</a>. In addition, we built a device in order to send and receive light signals from the bacterial culture. We have thus to create <strong>a means of communication from cell to machine and from machine to cell</strong>. For cell to machine communication, we chose to measure <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology">the red fluorescence of KillerRed</a>. A first function of our device is to excite and measure fluorescence intensity thanks to a light source, excitation/emission optics and a photodiode. In this way, our bacteria will be able to talk to our device. For machine to cell communication, we will use red light to activate <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology/KR"> light-inducible promoter</a> that triggers KillerRed production and white light to generate ROS thanks to KillerRed phototoxic activity. In our system, the rate of KillerRed production and the number of living cells will be controlled by the intensity of the red and white light beams. A second function of our device is therefore to generate controlled light intensities at different wavelengths. In this way, our device will be able to talk to our bacteria.</br></br></p>
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      <p>Our project aims to <strong>control the concentration of living bacteria in a culture</strong>. To do so, we designed <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology">a genetic network allowing light controlled cell growth</a>. In addition, we built a device in order to send and receive light signals from the bacterial culture. We have thus to create <strong>a means of communication from cell to machine and from machine to cell</strong>. For cell to machine communication, we chose to measure <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology">the red fluorescence of KillerRed</a>. The first function of our device is to excite and measure fluorescence intensity thanks to a light source, excitation/emission optics and a photodiode. In this way, our bacteria will be able to talk to our device. For machine to cell communication, we will use red light to activate <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology/KR"> light-inducible promoter</a> that triggers KillerRed production and white light to generate ROS thanks to KillerRed phototoxic activity. In our system, the rate of KillerRed production and the number of living cells will be controlled by the intensity of the red and white light beams. Therefore, a second function of our device is to generate controlled light intensities at different wavelengths. In this way, our device will be able to talk to our bacteria.</br></br></p>
                                               <p align="center"><img src="https://static.igem.org/mediawiki/2013/7/79/Logical_scheme.png" alt="Logical_sheme" width="500px" /></p>
                                               <p align="center"><img src="https://static.igem.org/mediawiki/2013/7/79/Logical_scheme.png" alt="Logical_sheme" width="500px" /></p>
                                               <p id="legend"><strong><em>Logical scheme of our device</em></strong></br>
                                               <p id="legend"><strong><em>Logical scheme of our device</em></strong></br>
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                                         The computer is giving orders to the microcontroller that control the light source (intensity and wavelengths) that illuminate the sample. Then the photodiode is measuring the level of fluorescence of the Red Fluorescent protein that is produced in the sample and the microcontroller is giving back the information to the computer that will change the intensity and the wavelength according to the measurement</br></br></p>
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                                         The computer gets information from the microcontroller about the fluorescence level of the bacterial suspension and calculates the intensity of the light to express KillerRed and control the cell density. The microcontroller itself controls the intensity and the spectrum of the light source that illuminates the sample. The photodiode measures the fluorescence level of KillerRed.</br></br></p>
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                                     <h2 id="Box">The Box</h2>
                                     <h2 id="Box">The Box</h2>
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                                       <p align="center"><object width="480" height="360"><param name="movie" value="//www.youtube.com/v/OY0-y8JZme0?version=3&amp;hl=fr_FR"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="//www.youtube.com/v/OY0-y8JZme0?version=3&amp;hl=fr_FR" type="application/x-shockwave-flash" width="480" height="360" allowscriptaccess="always" allowfullscreen="true"></embed></object></p>
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                                       <p align="center"><object width="480" height="360"><param name="movie" value="//www.youtube.com/v/OY0-y8JZme0?version=3&amp;hl=fr_FR"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="//www.youtube.com/v/OY0-y8JZme0?version=3&amp;hl=fr_FR" type="application/x-shockwave-flash" width="480" height="360" allowscriptaccess="always" allowfullscreen="true"></embed></object></br></br></p>
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                                       <p>Our device is built in such a way that the user <strong>just</strong> needs to <strong>input the concentration of living cells</strong> he wants and put <strong>the Erlenmeyer with our engineered bacteria</strong>. From that moment the device works in <strong>standalone mode</strong>. Firstly our device measures <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Fluo">the red fluorescence</a> of the culture that will be <strong>the baseline</strong>. Then it induces the <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology#KR">KillerRed</a> protein using <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology/KR">the red light inducible promoter</a>. Every <strong>5 minutes</strong>, Talk’E.Coli measures the level of red fluorescence of the culture and consequently of KillerRed. With <strong>these measurements and the parameters given by <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Parameters">the model</a></strong> it can calculate the moment when it needs to activate the ROS emissions with white light to stabilize the living cell concentration. The main asset of the device is that even if there is a lower increase of the concentration of KillerRed or if the killing rate is higher since it measures every 5 minutes the red fluorescence it can adjust exactly the light intensity to correct these rates.</br></br></p>
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                                       <p>Our device is built in such a way that the user <strong>only</strong> needs to <strong>define the concentration of living cells</strong> he wants and put <strong>the Erlenmeyer with our engineered bacteria</strong>. From that moment on, the device works in <strong>standalone manner</strong>. It first measures <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Fluo">the initial red fluorescence</a> (<strong>the baseline</strong>). Then it induces the <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology#KR">KillerRed</a> protein using <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology/KR">the red-inducible promoter</a>. Every <strong>5 minutes</strong>, TalkE'Coli measures <strong>the red fluorescence level</strong> of the culture. Then the light is switched on and the fluorescence is further recorded. This info is used to <strong>build <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Modelling/Building">a model</a> of cell growth and KillerRed response to illumination</strong>. The computer then calculates the <strong>time profile</strong> of the white light intensity used to <strong>stabilize</strong> the living cell concentration. <strong>The main asset</strong> of the device is that it recalculates the model <strong>during the run and thus adjusts the light intensity more precisely</strong>. Finally, when the living cell population is <strong>stabilized at 10% of its target value</strong>, the computer sends a signal to the user to tell him the success of the procedure.
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<p>Firstly we will explain the choice of the different components, then the several experiences we did to find the most accurate parameters for each part of the device : <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Photodiode">the photodiode and Arduino</a>, <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Fluo">fluorescence measurement</a>, <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Electronic">the electronic circuit</a>, <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Servo">the servomotor</a>. All these elements were then integrated in <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Box">the box</a> that we designed and built.</p>
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</br></br></p>
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<p>First, we will explain the choice of the different components, then the several experiments we did to find the most accurate parameters for each part of the device : <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Photodiode">the photodiode and Arduino</a>, <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Fluo">fluorescence measurement</a>, <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Electronic">the electronic circuit</a>, <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Servo">the servomotor</a>. All these elements were then integrated in <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo#Box">the box</a> that we designed and built.</p>
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