Difference between revisions of "Team:Lambert GA"

 
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<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Notebook">Notebook</a>
 
<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Notebook">Notebook</a>
 
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<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Model">Model</a>
 
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<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Results">Results</a><a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Demonstrate">Demonstrate</a>
 
<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Results">Results</a><a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Demonstrate">Demonstrate</a>
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<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Attributions">Attributions</a>
 
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<center> <img src="https://static.igem.org/mediawiki/2017/b/bd/T--Lambert_GA--gray_switch.png" style="width:45%; padding-top:80px;" align="middle">
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<center> <h2 class="maint"> Characterizing Non-Lysosomal Inducible Protein Degradation </h2></center>
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<h2> Characterization of Nonlysosomal Proteolysis </h2>
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<br><h3 style = "color: gray;" >Inspiration</h3>
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TinselPurple samples under varying levels of IPTG induction in the Chrome-Q base ready for imaging
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Scientists around the world struggle with questions regarding chromoprotein expression, specifically in regards to their use in biosensors.  Biosensors are often seen as low-cost alternatives to expensive detection equipment for underfunded labs and field work.  Lambert iGEM was inspired to address the problem of quantifying chromoprotein expression through development of a 3-D printed original device: Chrome-Q, software app, and verification with a chromoprotein genetic circuit. Additionally, samples were prepared with the 3-D Fuge (which was modified from Prakash Lab’s Paperfuge) to investigate the viability of using a low-cost centrifuge to process cells for visualization.<br>
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Quantifying color relies on measures of Red, Green, Blue (RGB) values and evaluating them in a color space known as Hue, Saturation and Value (HSV). Another consideration for color quantification is standardization of environmental light.  To achieve this a device, the Chrome-Q, was developed to create a standardized environment for capturing images using Android and Apple mobile device cameras.  Self-developed android software evaluates the RGB images resulting in HSV values.  These HSV values can be normalized for cell density through subsequent serial dilutions of the cultures and plating to count Colony Forming Units (CFU).<br><br>
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<i style="font-size: 14px; color: white;">Left: The HSL (hue, saturation and value) that the Chrome-Q measures
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Right: Chromoproteins under four different levels of IPTG induction plated to determine CFU's
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Throughout the development of the Chrome-Q, engineering design principles were implemented.  Feedback and resulting changes were implemented into the next iteration.  Five different prototypes led to the final development of two designs optimized for both Android and Apple mobile devices.  The Android software was written in C#.  The Chrome-Q stl files and software are available on the hardware page are open source.  <br>
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<p style="font-size: 20px; color: white; text-indent: 50px;">The device and software were then used to quantify data of ATUM’s Protein Paintbox proteins TinselPurple, ScroogeOrange and VirginiaViolet under varying levels of IPTG induction. An assembled genetic circuit of: Promoter- R0040, Ribosomal Binding Site B0034, and Tinsel purple (Tspurple)- (BBa_K1033906) (Uppsala 2013) was constructed to use in conjunction with a Protease mechanism of ClpXP.  ClpXP is a protein complex comprised of two parts.  ClpX recognizes an SsrA tag sequence at the end of a protein linearizes the tagged protein and brings it to ClpP.  ClpP is an ATPase and cleaves the primary peptide bonds resulting in degradation of the original protein into individual amino acids.  This demonstrated the potential usefulness of the 3-D fuge and Chrome-Q system as a replacement for fluorescent plate readers and centrifuges.<br>
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<i style="font-size: 14px; color: white;"> The full base of the Chrome-Q system with different dilutions of chromoproteins in the well plate
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<h2 style="margin-left: -300px; color: #7A7A79;"> Who We Are </h2><p style=" margin-left=300px; color: #7A7A79;">  We are comprised of 14 high school students from Suwanee, Georgia in the 10th, 11th, and 12th grades. We are an after-school/before-school club that meets simply for the love of synthetic biology.
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</p><a style="text-decoration: none;" href="https://2017.igem.org/Team:Lambert_GA/Team"><button class="button button1">Find Out More</button></a>
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Micronutrient deficiency is one of the leading causes of death in the world. According to the World Health Organization (WHO), more than 2 billion people - over 30% of the world’s population - suffer from micronutrient deficiencies today. In response, healthcare providers around the world have organized research facilities to diagnose and treat micronutrient deficiency. However, diagnosing these deficiencies has proved to be extremely expensive and, in many cases, very time consuming. To address this growing concern, medical researchers and synthetic biologists are creating simple and inexpensive “biosensors” that serve as handy diagnostic tests to use in the field to visualize micronutrient levels and determine whether the patient and population in general are lacking critical micronutrients in their diet. Despite these benefits, this versatile tool often yields inaccurate results, potentially leading to faulty diagnosis and the endangerment of an entire community.
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<div align="right" ><h2 class="smallt" style="color: #FFFFFF; margin-left: -91px;"> Our Project</h2></div>
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<p style=" margin-left=300px;color: white;"> As an underfunded lab, our project aimed to reduce costs of lab work. While characterizing non-lysosomal inducible protein degradation, we developed the Chrome-Q to quantify the degradation of protein.
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</p><a style="text-decoration: none;" href="https://2017.igem.org/Team:Lambert_GA/Description"><button class="button button2">Find Out More</button></a><br><br><br><br><br></div>
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<h2 style="margin=auto; margin-left=700px; color: #7A7A79;"> Human Practices </h2><p style=" margin-left=300px; color: #7A7A79;">  This past year, we have performed a variety of human practices, to educate the public about synthetic biology, as well as our project, through multiple outreach events.
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</p><a style="text-decoration: none;" href="https://2017.igem.org/Team:Lambert_GA/HP/Silver"><button class="button button1">Find Out More</button></a>
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<h2 style="color:#F19B45;"> Bronze </h2>
 
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<table>
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    <th width="20%">Requirements</th>
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    <th width="20%">Checklist</th>
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    <th width="60%">Explanation</th>
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    <td>Register and Attend</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/c/c6/Lambertigemcheckmarkgray.png" style="width:45px;"></td>
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    <td>Yes</td>
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  </tr>
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  <tr>
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    <td>Deliverable</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/b/bb/Lambertigemcheckmark.jpg" style="width:45px;"></td>
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    <td>Yes</td>
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    <td>Attributions</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/c/c6/Lambertigemcheckmarkgray.png" style="width:45px;"></td>
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    <td>Yes, click <a href="https://2017.igem.org/Team:Lambert_GA/Attributions">HERE</a> for more information.</td>
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    <td>Interlab Study</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/b/bb/Lambertigemcheckmark.jpg" style="width:45px;"></td>
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    <td>Yes, click <a href="https://2017.igem.org/Team:Lambert_GA/InterLab">HERE</a> for more information.</td>
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<h2 style="color:#c0c0c0;"> Silver </h2>
 
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With this in mind, the 2016 Lambert iGEM Team has focused primarily on enhancing existing biosensors and aiding in the diagnosis of micronutrient deficiencies throughout the world. We studied protein degradation and devised a “switch” to prevent the overexpression of specific reporters in biosensors in order to fine-tune biosensor accuracy. By using SsrA degradation tags, we characterized ClpXP, a protease involved in non-lysosomal proteolysis in many prokaryotes, to quantify the relative strength of degradation in GFP and purple chromoproteins.
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    <th width="20%">Requirements</th>
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    <th width="20%">Checklist</th>
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    <th width="60%">Explanation</th>
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    <td>Validated Part</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/c/c6/Lambertigemcheckmarkgray.png" style="width:45px;"></td>
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    <td>TsPurple and TsPurple with LAA degradation tag. <br> Click <a href="https://2017.igem.org/Team:Lambert_GA/Part_Collection">
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HERE</a> for more information regarding Lambert iGEM's parts.</td>
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    <td>Collaboration</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/b/bb/Lambertigemcheckmark.jpg" style="width:45px;"></td>
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    <td>Emory iGEM, UGA iGEM and TAS Taipei iGEM. <br> Click <a href="https://2017.igem.org/Team:Lambert_GA/Collaborations">HERE</a>
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for more information regarding Lambert iGEM's collaborations with other teams.</td>
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    <td>Human Practices</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/c/c6/Lambertigemcheckmarkgray.png" style="width:45px;"></td>
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    <td>Open House, Maker Faire, Survey, Ethics conferences. <br> Click <a href="https://2017.igem.org/Team:Lambert_GA/HP/Silver">HERE</a> for more information on Lambert iGEM's human practices.</td>
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<h2 style="color:#FFD700;"> Gold </h2>
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<p style=" margin-left=425px;"> Lorem ipsum dolor sit amet, consectetur adipiscing elit. Duis sed dolor sit amet quam sodales bibendum at ut tellus. Ut quis congue turpis, a semper ex. Praesent laoreet condimentum odio vitae bibendum. Proin rutrum vulputate felis, vitae volutpat nisi scelerisque eget.  Aliquam quis urna nisi. Curabitur id consectetur nulla, posuere malesuada orci. Donec rhoncus, nunc ac viverra ultricies, neque velit pulvinar ex, at efficitur enim arcu et leo. Ut hendrerit at dui a ornare. Integer fringilla purus in hendrerit molestie. Nunc diam ex, lacinia vitae eleifend ac, feugiat non dolor.
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<center>
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<table>
 +
  <tr>
 +
    <th width="20%">Requirements</th>
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    <th width="20%">Checklist</th>
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    <th width="60%">Explanation</th>
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  </tr>
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  <tr>
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    <td>Integrated Human Practices</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/c/c6/Lambertigemcheckmarkgray.png" style="width:45px;"></td>
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    <td><ul><li>Developed low cost solution for quantifying chromoprotein data </li>
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<li> Developed protocols to use a 3-D printed centrifuge for processing cells </li>
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<li> Gave feedback on 3-D fuge to Dr. Saad Bhamla of Georgia Institute of technology and Prakash Lab of Stanford. </li>
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<li> Donated blankets and supplies to impoverished families in Lambert iGEM's surrounding area. </li></ul>
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Click <a href="https://2017.igem.org/Team:Lambert_GA/HP/Gold_Integrated">HERE</a> for more information on Lambert iGEM's integrated human practices.
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</td>
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    <td>Improve BioBrick Part</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/b/bb/Lambertigemcheckmark.jpg" style="width:45px;"></td>
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    <td>The part this year's team improved upon is: <a href="http://parts.igem.org/Part:BBa_K1911001">BBa_K1911001</a> created by last year's Lambert iGEM team. The updated part this year is: <a href="http://parts.igem.org/wiki/index.php/Part:BBa_K1911001" style="color:#D49AE6;"> BBa_KK1911001: pLac-ClpXP-CI</a>, as we improved the characterization of last year's part. <br>
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Click <a href="https://2017.igem.org/Team:Lambert_GA/Updated_Part">HERE</a> for more information on Lambert iGEM's BioBrick Improvement of their project.
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</td>
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<tr>
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    <td>Model</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/c/c6/Lambertigemcheckmarkgray.png" style="width:45px;"></td>
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    <td><ul><li>Developed a working Chrome-Q and software app to quantify chromoprotein data</li>
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<li> Used a 3-D printed centrifuge to process cells and demonstrate viability in field work and underfunded labs.</li>
 +
<li> Developed designs for printing Chrome-Q device for both Android and Apple mobile devices.</li>
 +
<li> Developed protocols for obtaining and normalizing data.</li>
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<li> Developed protocols for sterilizing the Chrome-Q, base and 3-D fuge.</li></ul>
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Click <a href="https://2017.igem.org/Team:Lambert_GA/Model">HERE</a> for more information on Lambert iGEM's model in their project.
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</td>
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  <tr>
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    <td>Demonstration of Work</td>
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    <td><img src="https://static.igem.org/mediawiki/2017/b/bb/Lambertigemcheckmark.jpg" style="width:45px;"></td>
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    <td><ul><li>Developed a working Chrome-Q to quantify chromoprotein data in inducible genetic circuits.</li>
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<li> Developed a protocol in order to test construct at varying levels of IPTG and quantify data. </li>
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<li> Developed a software app to accurately analyze chromoprotein data from Chrome-Q.
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</ul>
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Click <a href="https://2017.igem.org/Team:Lambert_GA/Demonstrate">HERE</a> for information on Lambert iGEM's demonstration of their project.
  
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<h2 class="smallt" style="color: #FFFFFF;"> Medal Criteria</h2>
  
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<img id="imgcircle" src="https://www.nasa.gov/sites/default/files/thumbnails/image/hubble_friday_05132016.jpg">
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<h2 class="smallt" style="color: #FFFFFF;"> Award</h2>
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<img src="https://static.igem.org/mediawiki/2017/f/f2/Bronze.png"style="width:10%">
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<p style="color:white"> Bronze Medal</p>
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<a href="http://www.forsyth.k12.ga.us/lhs"><img src="https://static.igem.org/mediawiki/2017/3/36/RevisedLambertLonghorn.png" style="height:100px;padding:20px;" class="transparency"></a>
  
 
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Latest revision as of 01:21, 14 December 2017



Characterizing Non-Lysosomal Inducible Protein Degradation




TinselPurple samples under varying levels of IPTG induction in the Chrome-Q base ready for imaging


Scientists around the world struggle with questions regarding chromoprotein expression, specifically in regards to their use in biosensors. Biosensors are often seen as low-cost alternatives to expensive detection equipment for underfunded labs and field work. Lambert iGEM was inspired to address the problem of quantifying chromoprotein expression through development of a 3-D printed original device: Chrome-Q, software app, and verification with a chromoprotein genetic circuit. Additionally, samples were prepared with the 3-D Fuge (which was modified from Prakash Lab’s Paperfuge) to investigate the viability of using a low-cost centrifuge to process cells for visualization.

Quantifying color relies on measures of Red, Green, Blue (RGB) values and evaluating them in a color space known as Hue, Saturation and Value (HSV). Another consideration for color quantification is standardization of environmental light. To achieve this a device, the Chrome-Q, was developed to create a standardized environment for capturing images using Android and Apple mobile device cameras. Self-developed android software evaluates the RGB images resulting in HSV values. These HSV values can be normalized for cell density through subsequent serial dilutions of the cultures and plating to count Colony Forming Units (CFU).



Left: The HSL (hue, saturation and value) that the Chrome-Q measures
Right: Chromoproteins under four different levels of IPTG induction plated to determine CFU's

Throughout the development of the Chrome-Q, engineering design principles were implemented. Feedback and resulting changes were implemented into the next iteration. Five different prototypes led to the final development of two designs optimized for both Android and Apple mobile devices. The Android software was written in C#. The Chrome-Q stl files and software are available on the hardware page are open source.

The device and software were then used to quantify data of ATUM’s Protein Paintbox proteins TinselPurple, ScroogeOrange and VirginiaViolet under varying levels of IPTG induction. An assembled genetic circuit of: Promoter- R0040, Ribosomal Binding Site B0034, and Tinsel purple (Tspurple)- (BBa_K1033906) (Uppsala 2013) was constructed to use in conjunction with a Protease mechanism of ClpXP. ClpXP is a protein complex comprised of two parts. ClpX recognizes an SsrA tag sequence at the end of a protein linearizes the tagged protein and brings it to ClpP. ClpP is an ATPase and cleaves the primary peptide bonds resulting in degradation of the original protein into individual amino acids. This demonstrated the potential usefulness of the 3-D fuge and Chrome-Q system as a replacement for fluorescent plate readers and centrifuges.


The full base of the Chrome-Q system with different dilutions of chromoproteins in the well plate





Who We Are

We are comprised of 14 high school students from Suwanee, Georgia in the 10th, 11th, and 12th grades. We are an after-school/before-school club that meets simply for the love of synthetic biology.






Our Project

As an underfunded lab, our project aimed to reduce costs of lab work. While characterizing non-lysosomal inducible protein degradation, we developed the Chrome-Q to quantify the degradation of protein.








Human Practices

This past year, we have performed a variety of human practices, to educate the public about synthetic biology, as well as our project, through multiple outreach events.








Bronze


Requirements Checklist Explanation
Register and Attend Yes
Deliverable Yes
Attributions Yes, click HERE for more information.
Interlab Study Yes, click HERE for more information.


Silver


Requirements Checklist Explanation
Validated Part TsPurple and TsPurple with LAA degradation tag.
Click HERE for more information regarding Lambert iGEM's parts.
Collaboration Emory iGEM, UGA iGEM and TAS Taipei iGEM.
Click HERE for more information regarding Lambert iGEM's collaborations with other teams.
Human Practices Open House, Maker Faire, Survey, Ethics conferences.
Click HERE for more information on Lambert iGEM's human practices.


Gold


Requirements Checklist Explanation
Integrated Human Practices
  • Developed low cost solution for quantifying chromoprotein data
  • Developed protocols to use a 3-D printed centrifuge for processing cells
  • Gave feedback on 3-D fuge to Dr. Saad Bhamla of Georgia Institute of technology and Prakash Lab of Stanford.
  • Donated blankets and supplies to impoverished families in Lambert iGEM's surrounding area.
Click HERE for more information on Lambert iGEM's integrated human practices.
Improve BioBrick Part The part this year's team improved upon is: BBa_K1911001 created by last year's Lambert iGEM team. The updated part this year is: BBa_KK1911001: pLac-ClpXP-CI, as we improved the characterization of last year's part.
Click HERE for more information on Lambert iGEM's BioBrick Improvement of their project.
Model
  • Developed a working Chrome-Q and software app to quantify chromoprotein data
  • Used a 3-D printed centrifuge to process cells and demonstrate viability in field work and underfunded labs.
  • Developed designs for printing Chrome-Q device for both Android and Apple mobile devices.
  • Developed protocols for obtaining and normalizing data.
  • Developed protocols for sterilizing the Chrome-Q, base and 3-D fuge.
Click HERE for more information on Lambert iGEM's model in their project.
Demonstration of Work
  • Developed a working Chrome-Q to quantify chromoprotein data in inducible genetic circuits.
  • Developed a protocol in order to test construct at varying levels of IPTG and quantify data.
  • Developed a software app to accurately analyze chromoprotein data from Chrome-Q.
Click HERE for information on Lambert iGEM's demonstration of their project.

Medal Criteria



Award






Bronze Medal