Difference between revisions of "Team:US AFRL CarrollHS"

 
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{{US_AFRL_CarrollHS/WikiStripDown}} {{US_AFRL_CarrollHS/Layout!}} {{US_AFRL_CarrollHS/Bootstrap1}} {{US_AFRL_CarrollHS/Bootstrap2}} {{US_AFRL_CarrollHS/Bootstrap3}} {{US_AFRL_CarrollHS/Bootstrap4}} {{US_AFRL_CarrollHS/Bootstrap8}} {{US_AFRL_CarrollHS/navbar}} {{US_AFRL_CarrollHS/Slideshow}}  
  
 
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h1 {
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h1.right {
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<h1> Welcome to iGEM 2018! </h1>
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<p>Your team has been approved and you are ready to start the iGEM season! </p>
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<div class="column two_thirds_size"><h1> Project Description </h1> </div>
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<div class="column two_thirds_size"><p>Growing environmental concerns have led to an increase in the use of biofuels.  Biofuels contain higher concentrations of organic compounds, making them much more suitable for bacterial and fungal growth than traditional fuels. Consequently, fuel tanks or pipes containing biofuels are much more susceptible to biofilms. Biofilms cause a number of problems, including clogging pipes and filters, degrading the fuel, and corroding fuel tanks and pipes. The goal of this project is to combat biofilms by engineering an E. coli to swim to and destroy the biofilms.  </p>
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<p>Pseudomonas aeruginosa, a bacterium found in most biofilms, releases the quorum sensing molecule C4-Homoserine Lactone (C4-HSL). By sensing C4-HSL, the RhlR promoter will allow for the transcription of CheZ gene. Expression of the CheZ gene allows the flagella motors of E. coli to rotate counterclockwise in a straight-line path instead of tumbling. It continues to swim throughout the biofilm with the ability to tumble and redirect its path even if it leaves the concentration gradient of C4-HSL. Along with swimming to the biofilm, the engineered E. coli will produce chitinase and cinnamaldehyde to kill the fungi and bacteria.  Low concentrations of Cinnamaldehyde destroy bacteria by breaking down the cell membrane. However, fungi have cell walls, which prevents cinnamaldehyde from reaching the cell membrane.  Chitinase breaks down chitin, a major component of fungal cell walls, and allows the cinnamaldehyde to reach the cell membrane and kill fungal cells. Two forms of chitinase are used, Chitinase C-1 from Streptomyces griseus and Chitinase B4A from Serratia marcescens. Fusing chitinase and ice nucleation protein tethers the chitinase to the membrane of the engineered microbe, ensuring the chitinase will not diffuse away from the biofilm.  </p>
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<h1>Abstract</h1>
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<p>
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With growing environmental concerns, industries are increasingly relying on the use of biofuels. Biodiesel storage tanks are susceptible to water infiltration that often results in the formation of biofilms containing bacteria and fungi. Biofilms may clog pipes, degrade fuel, or corrode storage tanks. We set out to engineer a “seek, aim, and destroy” system for the remediation of microbial biofilm. <i>Pseudomonas aeruginosa</i>, commonly found in fuel biofilms, releases the quorum sensing molecule N-butanoyl-L-homoserine lactone (C4-HSL). Our engineered <i>E. coli</i> cells express CheZ protein in response to a concentration gradient of C4-HSL to activate the flagella motors and propel the cells towards the biofilm. In addition, the engineered <i>E. coli</i> expresses chitinase on its surface and secretes cinnamaldehyde. Chitinase breaks down chitin in the fungal cell walls, increasing the ability of cinnamaldehyde to destroy the fungi. Cinnamaldehyde also eliminates bacteria, thus remediating the biofilm.</p>
  
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<h1>Our Approach</h1>
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<p class="lead">We split our project up into three parts: Detect, Deliver, and Destroy</p>
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<p><b>Detect</b> C4-HSL, using the <i>RhlR</i> receptor gene.</p>
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<p><b>Deliver</b> the plasmid to the biofilm using <i>E. coli</i>'s natural chemotactic ability by expressing the protein CheZ</p>
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<p><b>Destroy</b> the bacterial and fungal biofilm by deploying two chitinase constructs to destroy chitin in the fungal cell walls, then utilizing cinnamaldehyde to kill the bacteria and fungi, which prevents biofilm formation.</p>
  
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<h3>Before you start</h3>
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<p> Please read the following pages:</p>
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<img src="https://static.igem.org/mediawiki/2018/b/b8/T--US_AFRL_CarrollHS--Circle1.jpeg">
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<ul>
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</div>
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<li>  <a href="https://2018.igem.org/Competition">Competition Hub</a> </li>
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<li> <a href="https://2018.igem.org/Competition/Deliverables/Wiki">Wiki Requirements page</a></li>
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<li> <a href="https://2018.igem.org/Resources/Template_Documentation">Template documentation</a></li>
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</ul>
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<img src="https://static.igem.org/mediawiki/2018/1/15/T--US_AFRL_CarrollHS--Circle2.jpeg">
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<div class="col-md-6">
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<h1 class="right">The Problem</h1>
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<p class="right2">There are three clear distinct layers that form in fuel containment systems: A water layer, the sludge layer, and the actual fuel layer. The formation of these layers usually takes a few weeks, but over time, water seeps into these underground tanks due to imperfections with storage techniques. With this water, microorganism contamination also comes into the mix. Now, a perfect environment for these microorganisms is present, because the water provides the perfect living conditions, and the biofuel contains high amounts of FAMEs (Fatty Acid Methyl Esters) for the microorganisms to feed on. Due to the density difference between fuel and water, the fuel sits on top, the water sits on the bottom, and the microorganisms sit in the middle forming the sludge, which is more commonly known as a biofilm.</p>
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<h3> Styling your wiki </h3>
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<p>You may style this page as you like or you can simply leave the style as it is. You can easily keep the styling and edit the content of these default wiki pages with your project information and completely fulfill the requirement to document your project.</p>
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<p>While you may not win Best Wiki with this styling, your team is still eligible for all other awards. This default wiki meets the requirements, it improves navigability and ease of use for visitors, and you should not feel it is necessary to style beyond what has been provided.</p>  
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<div class="row">
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<div class="col-md-6"> 
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<h1>Detect and Deliver</h1>
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<p>Our microbe will tumble through the fuel searching for C4-HSL, a molecule secreted by Pseudomonas aeruginosa, a bacteria commonly found in biofilms. Once C4-HSL is sensed, the protein CheZ is produced, which causes the microbe to move in a straight-line path. As higher concentrations of C4-HSL are found, the microbe moves closer to the biofilm. </p>
 
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<img src="https://static.igem.org/mediawiki/2018/6/6d/T--US_AFRL_CarrollHS--Circle3.jpeg">
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<img src="https://static.igem.org/mediawiki/2018/5/54/T--US_AFRL_CarrollHS--Circle4.jpeg">
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<div class="clear extra_space"></div>
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<div class="col-md-6">
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<h1 class="right">Destroy</h1>
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<p class="right2">After the engineered microbe is delivered to the biofilm, it must eliminate the fungal and bacterial contaminants present there. Cinnamaldehyde is used in order to permeate through the cell membrane since it is small enough to enter the phospholipid bilayer. However, fungi has additional protection cell wall that must be punctured to allow access to the membrane. The cell wall is anchored to the membrane by a polymer called chitin, so an additional enzyme called chitinase was used to open up the cell wall.</p>
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<h1>Solution</h1>
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<h3> Uploading pictures and files </h3>
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<p>Once our three steps have been implemented, the engineered microbe will be dispersed through the biofilm. Theoretically, the cinnamaldehyde will kill all living microorganisms in the biofilm, including the engineered microbe. Thus, the biofilm will effectively be destroyed.</p>
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<p> You must upload any pictures and files to the iGEM 2018 server. Remember to keep all your pictures and files within your team's namespace or at least include your team's name in the file name. </p>
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<p>When you upload, set the "Destination Filename" to <b> T--YourOfficialTeamName--NameOfFile.jpg</b>. (If you don't do this, someone else might upload a different file with the same "Destination Filename", and your file would be erased!)</p>
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<a href="https://2018.igem.org/Special:Upload">
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UPLOAD FILES
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<img src="https://static.igem.org/mediawiki/2018/6/63/T--US_AFRL_CarrollHS--Circle5.jpeg">
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<h3> Wiki template information </h3>
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<p>We have created these wiki template pages to help you get started and to help you think about how your team will be evaluated. You can find a list of all the pages tied to awards here at the <a href="https://2018.igem.org/Judging/Pages_for_Awards">Pages for awards</a> link. You must edit these pages to be evaluated for medals and awards, but ultimately the design, layout, style and all other elements of your team wiki is up to you!</p>
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<h3> Editing your wiki </h3>
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<p>On this page you can document your project, introduce your team members, document your progress and share your iGEM experience with the rest of the world! </p>
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<p>Use WikiTools - Edit in the black menu bar to edit this page</p>
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  <a href="https://2018.igem.org/wiki/index.php?title=Team:US_AFRL_CarrollHS&action=edit">
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EDIT PAGE
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<img src="https://static.igem.org/mediawiki/2018/6/68/T--US_AFRL_CarrollHS--Circle6.jpeg">
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<h1 class="right">Result</h1>
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<p class="right2">With the sludge layer removed, biofuels will be a much more viable option for the consumer. With biofilms removed, fouling, degradation, and corrosion will be effectively eliminated from the storage process. This allows biofuels to be used more regularly resulting in a cheaper and healthier fuel for the world today.</p>
 
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<h1>Our Music Video!</h1>
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<p>We had a lot of fun working on our iGEM project, including the creation of our music video.</p>
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<h3>Tips</h3>
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<p>This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started: </p>
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<li>State your accomplishments! Tell people what you have achieved from the start. </li>
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<li>Be clear about what you are doing and how you plan to do this.</li>
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<li>You have a global audience! Consider the different backgrounds that your users come from.</li>
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<li>Make sure information is easy to find; nothing should be more than 3 clicks away.  </li>
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<li>Avoid using very small fonts and low contrast colors; information should be easy to read.  </li>
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<li>Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the <a href="https://2018.igem.org/Calendar">iGEM 2018 calendar</a> </li>
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<li>Have lots of fun! </li>
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<video width="100%" height="500" controls>  
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<source src="https://static.igem.org/mediawiki/2018/8/80/T--US_AFRL_CarrollHS--MusicVideo.mp4"type="video/mp4">  
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<h3>Inspiration</h3>
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</video>
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<p> You can also view other team wikis for inspiration! Here are some examples:</p>
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<li> <a href="https://2014.igem.org/Team:SDU-Denmark/"> 2014 SDU Denmark </a> </li>
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<li> <a href="https://2014.igem.org/Team:Aalto-Helsinki">2014 Aalto-Helsinki</a> </li>
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<li> <a href="https://2014.igem.org/Team:LMU-Munich">2014 LMU-Munich</a> </li>
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<li> <a href="https://2014.igem.org/Team:Michigan"> 2014 Michigan</a></li>
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<li> <a href="https://2014.igem.org/Team:ITESM-Guadalajara">2014 ITESM-Guadalajara </a></li>
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<li> <a href="https://2014.igem.org/Team:SCU-China"> 2014 SCU-China </a></li>
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{{US_AFRL_CarrollHS/footer}}

Latest revision as of 02:02, 18 October 2018

Abstract

With growing environmental concerns, industries are increasingly relying on the use of biofuels. Biodiesel storage tanks are susceptible to water infiltration that often results in the formation of biofilms containing bacteria and fungi. Biofilms may clog pipes, degrade fuel, or corrode storage tanks. We set out to engineer a “seek, aim, and destroy” system for the remediation of microbial biofilm. Pseudomonas aeruginosa, commonly found in fuel biofilms, releases the quorum sensing molecule N-butanoyl-L-homoserine lactone (C4-HSL). Our engineered E. coli cells express CheZ protein in response to a concentration gradient of C4-HSL to activate the flagella motors and propel the cells towards the biofilm. In addition, the engineered E. coli expresses chitinase on its surface and secretes cinnamaldehyde. Chitinase breaks down chitin in the fungal cell walls, increasing the ability of cinnamaldehyde to destroy the fungi. Cinnamaldehyde also eliminates bacteria, thus remediating the biofilm.

Our Approach

We split our project up into three parts: Detect, Deliver, and Destroy

Detect C4-HSL, using the RhlR receptor gene.

Deliver the plasmid to the biofilm using E. coli's natural chemotactic ability by expressing the protein CheZ

Destroy the bacterial and fungal biofilm by deploying two chitinase constructs to destroy chitin in the fungal cell walls, then utilizing cinnamaldehyde to kill the bacteria and fungi, which prevents biofilm formation.

The Problem

There are three clear distinct layers that form in fuel containment systems: A water layer, the sludge layer, and the actual fuel layer. The formation of these layers usually takes a few weeks, but over time, water seeps into these underground tanks due to imperfections with storage techniques. With this water, microorganism contamination also comes into the mix. Now, a perfect environment for these microorganisms is present, because the water provides the perfect living conditions, and the biofuel contains high amounts of FAMEs (Fatty Acid Methyl Esters) for the microorganisms to feed on. Due to the density difference between fuel and water, the fuel sits on top, the water sits on the bottom, and the microorganisms sit in the middle forming the sludge, which is more commonly known as a biofilm.

Detect and Deliver

Our microbe will tumble through the fuel searching for C4-HSL, a molecule secreted by Pseudomonas aeruginosa, a bacteria commonly found in biofilms. Once C4-HSL is sensed, the protein CheZ is produced, which causes the microbe to move in a straight-line path. As higher concentrations of C4-HSL are found, the microbe moves closer to the biofilm.

Destroy

After the engineered microbe is delivered to the biofilm, it must eliminate the fungal and bacterial contaminants present there. Cinnamaldehyde is used in order to permeate through the cell membrane since it is small enough to enter the phospholipid bilayer. However, fungi has additional protection cell wall that must be punctured to allow access to the membrane. The cell wall is anchored to the membrane by a polymer called chitin, so an additional enzyme called chitinase was used to open up the cell wall.

Solution

Once our three steps have been implemented, the engineered microbe will be dispersed through the biofilm. Theoretically, the cinnamaldehyde will kill all living microorganisms in the biofilm, including the engineered microbe. Thus, the biofilm will effectively be destroyed.

Result

With the sludge layer removed, biofuels will be a much more viable option for the consumer. With biofilms removed, fouling, degradation, and corrosion will be effectively eliminated from the storage process. This allows biofuels to be used more regularly resulting in a cheaper and healthier fuel for the world today.

Our Music Video!

We had a lot of fun working on our iGEM project, including the creation of our music video.

Contact us @iGEMLabPats or iGEM@carrollhs.org