Difference between revisions of "Team:Calgary/Human Practices"
18desilvni (Talk | contribs) |
|||
| Line 1: | Line 1: | ||
| − | {{Calgary/Imposter-Navbar}} | + | {{Calgary/Imposter-Navbar-No-Logo}} |
<!-- | <!-- | ||
Final Colours: | Final Colours: | ||
| Line 9: | Line 9: | ||
light red: #FBF6F3 | light red: #FBF6F3 | ||
light yellow: #F1C23F (jumbotron h1) | light yellow: #F1C23F (jumbotron h1) | ||
| + | --> | ||
| + | <!-- INSTRUCTIONS for adding more sections | ||
| + | |||
| + | 1. copy and paste the following code above the message at end of code (message says CONTENT): | ||
| + | |||
| + | <div class = "NAME" id="NAME"> | ||
| + | <h2>NAME</h2> | ||
| + | <h4>HEADER</h4> | ||
| + | <p> | ||
| + | PASTE IN TEXT HERE | ||
| + | </p> | ||
| + | </div> | ||
| + | <br> | ||
| + | <hr> | ||
| + | |||
| + | 2. Where it says NAME, type in name of section. This has to be the SAME thing you type in for every place it says NAME. for the NAME after the id, you cannot include spaces between words, so use dashes instead, also don't use capitals. (Ex. NAME of section is "Future Directions", so i would paste that in where ever it says NAME. for the NAME after the id, I would type in "future-directions".) Also type in desired HEADER and text where it says PASTE IN TEXT HERE. | ||
| + | |||
| + | 3. Now we have to add the section to the sidebar menu. For this, go through the code and find the part where it says SIDEBAR. where it says "replace-title" paste in the NAME you put in after the id (Ex. I would paste in "future-directions".) Where it says "Title" paste in the NAME of the section. | ||
| + | |||
| + | 4. Now go to the part of the code where it says "References section of page slightly above the header". In the line of code after that, add in #NAME after a comma after the last one that is there before the curly brackets. (Ex. I would paste in #future-directions after the last item in that line of code after a comma.) | ||
| + | |||
| + | 5. I've included the example in the code so you can follow along.That's all. You're all done!! :D If you need help just ask! | ||
| + | --> | ||
| + | <!-- INSTRUCTIONS for adding more experiment sections to accordion: | ||
| + | |||
| + | 1. copy and paste the following above the message showing where to paste (it says ACCORDION in the message): | ||
| + | |||
| + | <div class="card"> | ||
| + | <div class="card-header" id="headingREPLACE"> | ||
| + | <h2 class="mb-0"> | ||
| + | <button class="btn btn-link collapsed" type="button" data-toggle="collapse" data-target="#collapseREPLACE" aria-expanded="false" aria-controls="collapseREPLACE"> | ||
| + | EXPERIMENT | ||
| + | </button> | ||
| + | </h2> | ||
| + | </div> | ||
| + | <div id="collapseREPLACE" class="collapse" aria-labelledby="headingREPLACE" data-parent="#accordionExample"> | ||
| + | <div class="card-body"> | ||
| + | PASTE IN TEXT HERE. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | 2. Every place where it says REPLACE in the code, type in the next highest numerical number. (Ex. if I am putting in a new experiment I would copy and paste the code above, and type in "Five" where REPLACE is because the last number used in the code was "Four".) The main thing is that whatever you type in for REPLACE has to only be present there and nowhere else in the code!!!! | ||
| + | |||
| + | 3. You can now copy and paste in your text from your protocol where it says PASTE IN TEXT HERE. And paste in the name of your experiment where it says EXPERIMENT. | ||
| + | |||
--> | --> | ||
<html> | <html> | ||
<head> | <head> | ||
| + | <!-- LINKS THE SIDEBAR CSS SHEET BECAUSE THIS PAGE USES A SIDEBAR --> | ||
| + | <link | ||
| + | rel="stylesheet" | ||
| + | type="text/css" href="https://2020.igem.org/Template:Calgary/Imposter-Sidebar-2-Style?action=raw&ctype=text/css" | ||
| + | /> | ||
<link href="https://fonts.googleapis.com/css2?family=Public+Sans:wght@500;600;700&display=swap" rel="stylesheet"> | <link href="https://fonts.googleapis.com/css2?family=Public+Sans:wght@500;600;700&display=swap" rel="stylesheet"> | ||
| Line 18: | Line 69: | ||
rel="stylesheet" | rel="stylesheet" | ||
type="text/css" | type="text/css" | ||
| − | href="https://2020.igem.org/Template:Calgary/Imposter- | + | href="https://2020.igem.org/Template:Calgary/Imposter-Content-Style?action=raw&ctype=text/css" |
/> | /> | ||
| Line 24: | Line 75: | ||
<script src="https://2020.igem.org/Template:Calgary/DynamicSizeModelPage?action=raw&ctype=text/javascript"></script> | <script src="https://2020.igem.org/Template:Calgary/DynamicSizeModelPage?action=raw&ctype=text/javascript"></script> | ||
<style> | <style> | ||
| + | |||
/* The banner image */ | /* The banner image */ | ||
.banner-image { | .banner-image { | ||
/* Use "linear-gradient" to add a darken background effect to the image (photographer.jpg). This will make the text easier to read */ | /* Use "linear-gradient" to add a darken background effect to the image (photographer.jpg). This will make the text easier to read */ | ||
| − | background-image: linear-gradient(to bottom, rgba(0,0,0,0.5) 0%,rgba(0,0,0,0.5) 100%), url("https://static.igem.org/mediawiki/2020/ | + | background-image: linear-gradient(to bottom, rgba(0,0,0,0.5) 0%,rgba(0,0,0,0.5) 100%), url("https://static.igem.org/mediawiki/2020/4/43/T--Calgary--simran.jpg"); |
| − | + | background-position: 0px -200px; | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | background-position: | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
} | } | ||
| − | |||
| Line 56: | Line 98: | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | /* | + | /* Fixing the top content under the navbar */ |
| − | . | + | .banner-image { |
| − | + | position: relative; | |
| − | + | } | |
| − | + | ||
| − | } | + | |
| − | |||
| Line 95: | Line 123: | ||
.btn {color:#882916;} | .btn {color:#882916;} | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
</style> | </style> | ||
</head> | </head> | ||
| − | <body style="background-color: #FFFFFF;" | + | |
| − | + | <body style="background-color: #FFFFFF;" ;> | |
<div class="container-fluid"> | <div class="container-fluid"> | ||
| − | + | <!-- SIDEBAR --> | |
| + | <div class="sidenav" id="sidenav"> | ||
| + | <a href="https://2020.igem.org/Team:Calgary" | ||
| + | ><img | ||
| + | src="https://static.igem.org/mediawiki/2020/c/c1/T--Calgary--whitelogo.jpg" | ||
| + | style="height: 150px; width: auto; padding-top:20px; position:relative; left:-20px;" | ||
| + | /></a> | ||
| + | |||
| + | <a href="#project-design">Project Designs</a> | ||
| + | <br> | ||
| + | <a href="#part-design">Part Design</a> | ||
| + | <br> | ||
| + | <a href="#experimental-design">Experimental Design</a> | ||
| + | <br> | ||
| + | <a href="#future-directions">Future Directions</a> | ||
| + | <br> | ||
| + | <a href="#understanding-the-problem">Understanding the Problem</a> | ||
| + | <br> | ||
| + | <a href="#experimental-troubleshooting">Experimental Troubleshooting</a> | ||
| + | |||
| + | <br> | ||
| + | <a href="#expected-results">Expected Results</a> | ||
| + | <br> | ||
| + | <a href="#references"> References</a> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | <div class="main"> | ||
<!-- HEADER/BANNER --> | <!-- HEADER/BANNER --> | ||
<div class="banner-image"> | <div class="banner-image"> | ||
<div class="banner-text resp"> | <div class="banner-text resp"> | ||
| − | + | <h1 style="font-family: 'Public Sans', sans-serif; font-weight: 600;">ANTHELMINTIC: THYMOL</h1> | |
| + | |||
</div> | </div> | ||
</div> | </div> | ||
<!-- BODY --> | <!-- BODY --> | ||
| − | <div | + | <div class="intro-body"> |
| − | <div | + | <div class = "project-design" id="project-design"> |
| − | < | + | <h2>OVERVIEW</h2> |
| − | < | + | <img class="img-fluid"style="width:100%;"src="https://static.igem.org/mediawiki/2020/5/5a/T--Calgary--thymolpipeline.png" /> |
| + | <br><br><br> | ||
| + | |||
| + | <h4>Optimizing Nutrient Absorption Through Anthelmintics </h4> | ||
<p> | <p> | ||
| − | + | ||
| + | Mass-supplementation of Vitamin A to deficient regions have been ongoing for decades.However, even with biannual vitamin A supplementation for children and biofortified crops such as golden rice exist, intestinal parasites still play the villain in preventing the vitamin’s assimilation by perforating the intestines. Organizations such as the UN, WHO, and the Bill Gates Foundation couple biannual vitamin supplementation with deworming, but lack of clean water and proper footwear allow these parasites to return and thrive in the intestines. The Oviita system sustainably supplements the consumer with vitamin A and thymol, an anthelmintic agent which is proven to work against worms and their larvae. By doing so, the intestines can recover and maximize absorption of vitamin A in the body. | ||
| + | |||
</p> | </p> | ||
| + | <img class="img-fluid"src="https://static.igem.org/mediawiki/2020/9/99/T--Calgary--thymoloverview.jpeg"> | ||
| + | <br> | ||
| − | + | </div> | |
| − | + | ||
| − | + | ||
| + | <br> | ||
| + | <hr> | ||
| + | <div class = "understanding-the-problem" id="understanding-the-problem"> | ||
| + | <h2>UNDERSTANDING THE PROBLEM</h2> | ||
| + | <h4>Who we talked to</h4> | ||
| + | <p> | ||
| + | To understand the extent of the problem, we talked to people who work directly with the problem. | ||
| + | </p> | ||
| + | <img class="img-fluid"src="https://static.igem.org/mediawiki/2020/4/47/T--Calgary--ThymolHP.jpeg"> | ||
| − | < | + | <p> |
| − | + | <ul> | |
| + | <li>Dr. Sanou Dia - Even with VA supplies, intestinal parasites are a big problem. Lack of footwear and clean water cause them to return, and hinder vitamin absorption. </li> | ||
| + | <li>Dr. Paul E. Mains - There is merit to producing thymol as worms and intestinal parasites may also develop resistance to anthelmintic drugs. </li> | ||
| + | <li> Lourlin Ugdimay - Something about nutrition deficiencies and how they are distributed, maybe even purga. </li> | ||
| + | <li> Dr. John Gileard - Parasites are not only problematic in humans, but also livestock. It is important to address this because people from vulnerable areas rely on their livestock for food and income, and there are only 2 main anthelmintic drugs used in the industry, and snps can easily happen. </li> | ||
| + | </ul> | ||
| + | |||
| + | <br> | ||
| + | After our conversations with our HP contacts, we realized that we had to do something about the problem. Since our yeast is meant to be consumed as a nutritional supplement, we need a compound that is a naturally-occuring anthelmintic that is safe for consumption. [insert transition to thymol]. | ||
| + | |||
| + | <br><br> | ||
| + | <b>Thymol</b> is a monoterpenoid found in thyme leaves (<i>Thymus vulgaris</i>). It has long been used in traditional medicine for its antioxidant, anti-inflammatory, and antihyperlipidemic properties (Meeran et al. 2017). Although thymol is “Generally Recognized As Safe” (GRAS) by the United States Food and Drug Administration and may be ingested with “negligible toxicity”, extreme doses may induce unwanted side effects such burning pain in the oesophagus, nausea, abdominal pain, vomiting, and dizziness among others (CITE: SCBT Datasheets). This compound is normally administered through essential oils, tablets, or used as a disinfectant by integration in soaps. | ||
| + | |||
| + | <br><br> | ||
| + | INSERT study about thymol pharmacokinetics. And how oil form is absorbed more quickly because thymol is lipophylic, so yarrowia is a good chassis! | ||
| + | |||
| + | <br><br> | ||
| + | Since Y. lipolytica already has the MEP pathway and produces GPP we will only need the addition of two genes: a terpene synthase (TvTPS1) and a cytochrome P450 (CYP71D178). | ||
| + | |||
| + | </p> | ||
| + | </div> | ||
| + | <br> | ||
| + | |||
| + | |||
| + | <br> | ||
| + | <hr> | ||
| + | <div class = "part-design" id="part-design"> | ||
| + | <h2>PART DESIGN</h2> | ||
| + | <h4>Thoughtful design of genetic constructs</h4> | ||
| + | <p> | ||
| + | Thymol parts | ||
| + | </p> | ||
| + | </div> | ||
| + | <br> | ||
| + | <hr> | ||
| + | <div class = "experimental-design" id="experimental-design"> | ||
| + | <h2>EXPERIMENTAL DESIGN</h2> </div> | ||
| + | <h4>Thoughtful design of experiments</h4> | ||
| + | <p> | ||
| + | [Insert thymol lab workflow graphic here] | ||
| + | </p> | ||
| + | <!--ACCORDION --> | ||
| + | |||
| + | <div class="accordion" id="accordionExample"> | ||
| + | <div class="card"> | ||
| + | <div class="card-header" id="headingOne"> | ||
| + | <h2 class="mb-0"> | ||
| + | <button class="btn btn-link" type="button" data-toggle="collapse" data-target="#collapseOne" aria-expanded="true" aria-controls="collapseOne"> | ||
| + | Cloning | ||
| + | </button> | ||
| + | </h2> | ||
</div> | </div> | ||
| − | |||
| − | < | + | <div id="collapseOne" class="collapse show" aria-labelledby="headingOne" data-parent="#accordionExample"> |
| + | <div class="card-body"> | ||
| + | To express thymol in Yarrowia lipolytica a terpene synthase (TvTPS1) and a cytochrome P450 (CYP71D178) will be introduced. To keep substantial DNA stocks we will first transform our constructs in pSB1A3 into chemically competent Escherichia coli DH5α. This DNA would then be extracted through plasmid miniprep. Since Y. lipolytica functions best with non-homologous end joining, we will linearize the plasmid from E. coli with (insert restriction enzyme here) then transform it into Y. lipolytica using lithium acetate transformation which will result in random integration into its genome. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="card"> | ||
| + | <div class="card-header" id="headingTwo"> | ||
| + | <h2 class="mb-0"> | ||
| + | <button class="btn btn-link collapsed" type="button" data-toggle="collapse" data-target="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo"> | ||
| + | CONFIRMATION OF THYMOL PRODUCTION/ ENZYME CHARACTERIZATION | ||
| + | </button> | ||
| + | </h2> | ||
| + | </div> | ||
| + | <div id="collapseTwo" class="collapse" aria-labelledby="headingTwo" data-parent="#accordionExample"> | ||
| + | <div class="card-body"> | ||
| + | Can still express in E. coli even though it’s codon-optimized for Yarrowia lipolytica. | ||
| + | Ni-NTA purification | ||
| + | Western Blot | ||
| + | Bradford Assay | ||
| − | + | TvTPS1 | |
| − | + | To characterize the TvTPS1 gene, just add GPP. | |
| + | Then check absorbance values ? | ||
| + | |||
| + | CYP71D178 | ||
| + | Gamma-Terpinene. | ||
| + | |||
| + | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="card"> | |
| − | + | <div class="card-header" id="headingThree"> | |
| + | <h2 class="mb-0"> | ||
| + | <button class="btn btn-link collapsed" type="button" data-toggle="collapse" data-target="#collapseThree" aria-expanded="false" aria-controls="collapseThree"> | ||
| + | CAENORHABDITIS ELEGANS TESTING | ||
| + | </button> | ||
| + | </h2> | ||
</div> | </div> | ||
| + | <div id="collapseThree" class="collapse" aria-labelledby="headingThree" data-parent="#accordionExample"> | ||
| + | <div class="card-body"> | ||
| + | Caenorhabditis elegans is the gold-standard for testing anthelmintic drugs extensively before its clinical trials in humans. To prove thymol’s activity we will be testing its effects on C. elegans by soaking Escherichia coli in varying thymol concentrations, then feed it to the C. elegans. (Observe movement for four days. ). Need to know which concentrations are lethal, how much our | ||
| + | |||
| + | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| + | <div class="card"> | ||
| + | <div class="card-header" id="headingFour"> | ||
| + | <h2 class="mb-0"> | ||
| + | <button class="btn btn-link collapsed" type="button" data-toggle="collapse" data-target="#collapseFour" aria-expanded="false" aria-controls="collapseFour"> | ||
| + | Experiment 4 | ||
| + | </button> | ||
| + | </h2> | ||
| + | </div> | ||
| + | <div id="collapseFour" class="collapse" aria-labelledby="headingFour" data-parent="#accordionExample"> | ||
| + | <div class="card-body"> | ||
| + | Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| − | |||
| − | |||
| + | </div> <!--Everything above this is in the ACCORDION. So copy and paste a new experiment above here. --> | ||
| + | <br> | ||
| + | <hr> | ||
| + | <div class = "expected-results" id="expected-results"> | ||
| + | <h2>Expected Results</h2> | ||
| + | <h4>For when we get back to the lab</h4> | ||
| − | + | <p> | |
| + | In order to provide a sustainable, community-based solution, we plan | ||
| + | to genetically modify <i>Rhodosporidium toruloides</i>, an oleaginous | ||
| + | yeast that naturally produces beta-carotene and lipids, to be more | ||
| + | robust and resource-efficient. By modifying the yeast to produce | ||
| + | cellulase, it can then use common agricultural waste products as an | ||
| + | energy source for synthesizing its oil. It can then be eaten as a | ||
| + | vitamin A supplement. The yeast strain, while naturally safe and | ||
| + | non-pathogenic, will also be genetically modified to include a kill | ||
| + | switch for bio-containment, and optimized for oil production. | ||
| + | </p> | ||
| + | <!--ACCORDION --> | ||
| + | <div class="accordion" id="accordionExample"> | ||
| + | <div class="card"> | ||
| + | <div class="card-header" id="headingOne"> | ||
| + | <h2 class="mb-0"> | ||
| + | <button class="btn btn-link" type="button" data-toggle="collapse" data-target="#collapseOne" aria-expanded="true" aria-controls="collapseOne"> | ||
| + | Cloning | ||
| + | </button> | ||
| + | </h2> | ||
| + | </div> | ||
| + | <div id="collapseOne" class="collapse show" aria-labelledby="headingOne" data-parent="#accordionExample"> | ||
| + | <div class="card-body"> | ||
| + | To express thymol in Yarrowia lipolytica a terpene synthase (TvTPS1) and a cytochrome P450 (CYP71D178) will be introduced. To keep substantial DNA stocks we will first transform our constructs in pSB1A3 into chemically competent Escherichia coli DH5α. This DNA would then be extracted through plasmid miniprep. Since Y. lipolytica functions best with non-homologous end joining, we will linearize the plasmid from E. coli with (insert restriction enzyme here) then transform it into Y. lipolytica using lithium acetate transformation which will result in random integration into its genome. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="card"> | ||
| + | <div class="card-header" id="headingTwo"> | ||
| + | <h2 class="mb-0"> | ||
| + | <button class="btn btn-link collapsed" type="button" data-toggle="collapse" data-target="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo"> | ||
| + | CONFIRMATION OF THYMOL PRODUCTION/ ENZYME CHARACTERIZATION | ||
| + | </button> | ||
| + | </h2> | ||
| + | </div> | ||
| + | <div id="collapseTwo" class="collapse" aria-labelledby="headingTwo" data-parent="#accordionExample"> | ||
| + | <div class="card-body"> | ||
| + | Can still express in E. coli even though it’s codon-optimized for Yarrowia lipolytica. | ||
| + | Ni-NTA purification | ||
| + | Western Blot | ||
| + | Bradford Assay | ||
| + | TvTPS1 | ||
| + | To characterize the TvTPS1 gene, just add GPP. | ||
| + | Then check absorbance values ? | ||
| + | |||
| + | CYP71D178 | ||
| + | Gamma-Terpinene. | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="card"> | ||
| + | <div class="card-header" id="headingThree"> | ||
| + | <h2 class="mb-0"> | ||
| + | <button class="btn btn-link collapsed" type="button" data-toggle="collapse" data-target="#collapseThree" aria-expanded="false" aria-controls="collapseThree"> | ||
| + | CAENORHABDITIS ELEGANS TESTING | ||
| + | </button> | ||
| + | </h2> | ||
| + | </div> | ||
| + | <div id="collapseThree" class="collapse" aria-labelledby="headingThree" data-parent="#accordionExample"> | ||
| + | <div class="card-body"> | ||
| + | Caenorhabditis elegans is the gold-standard for testing anthelmintic drugs extensively before its clinical trials in humans. To prove thymol’s activity we will be testing its effects on C. elegans by soaking Escherichia coli in varying thymol concentrations, then feed it to the C. elegans. (Observe movement for four days. ). Need to know which concentrations are lethal, how much our | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="card"> | ||
| + | <div class="card-header" id="headingFour"> | ||
| + | <h2 class="mb-0"> | ||
| + | <button class="btn btn-link collapsed" type="button" data-toggle="collapse" data-target="#collapseFour" aria-expanded="false" aria-controls="collapseFour"> | ||
| + | Western Blot | ||
| + | </button> | ||
| + | </h2> | ||
| + | </div> | ||
| + | <div id="collapseFour" class="collapse" aria-labelledby="headingFour" data-parent="#accordionExample"> | ||
| + | <div class="card-body"> | ||
| + | Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | </div> <!--Everything above this is in the ACCORDION. So copy and paste a new experiment above here. --> | ||
</div> | </div> | ||
| + | <hr> | ||
| + | <div class = "Future Directions" id="future-directions"> | ||
| + | <h2>FUTURE DIRECTIONS</h2> | ||
| + | <h4>Next Steps</h4> | ||
| + | <p> | ||
| + | In order to provide a sustainable, community-based solution, we plan | ||
| + | to genetically modify <i>Rhodosporidium toruloides</i>, an oleaginous | ||
| + | yeast that naturally produces beta-carotene and lipids, to be more | ||
| + | robust and resource-efficient. By modifying the yeast to produce | ||
| + | cellulase, it can then use common agricultural waste products as an | ||
| + | energy source for synthesizing its oil. It can then be eaten as a | ||
| + | vitamin A supplement. The yeast strain, while naturally safe and | ||
| + | non-pathogenic, will also be genetically modified to include a kill | ||
| + | switch for bio-containment, and optimized for oil production. | ||
| + | </p> | ||
| + | </div> | ||
| + | <br> | ||
| + | <hr> | ||
| + | |||
| + | |||
| + | <br> | ||
| + | <hr> | ||
| + | <div class = "understanding-the-problem" id="understanding-the-problem"> | ||
| + | <h2>References</h2> | ||
| + | <p> | ||
| + | Paper 1 | ||
| + | |||
| + | </p> | ||
| + | |||
| + | </div> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
</div> <!-- add in new section of CONTENT above this --> | </div> <!-- add in new section of CONTENT above this --> | ||
</div> | </div> | ||
| − | + | </div> | |
</body> | </body> | ||
</html> | </html> | ||
{{Calgary/Imposter-Footer}} | {{Calgary/Imposter-Footer}} | ||
Revision as of 04:41, 20 October 2020
Project Designs
Part Design
Experimental Design
Future Directions
Understanding the Problem
Experimental Troubleshooting
Expected Results
References
OVERVIEW
Optimizing Nutrient Absorption Through Anthelmintics
Mass-supplementation of Vitamin A to deficient regions have been ongoing for decades.However, even with biannual vitamin A supplementation for children and biofortified crops such as golden rice exist, intestinal parasites still play the villain in preventing the vitamin’s assimilation by perforating the intestines. Organizations such as the UN, WHO, and the Bill Gates Foundation couple biannual vitamin supplementation with deworming, but lack of clean water and proper footwear allow these parasites to return and thrive in the intestines. The Oviita system sustainably supplements the consumer with vitamin A and thymol, an anthelmintic agent which is proven to work against worms and their larvae. By doing so, the intestines can recover and maximize absorption of vitamin A in the body.
UNDERSTANDING THE PROBLEM
Who we talked to
To understand the extent of the problem, we talked to people who work directly with the problem.
- Dr. Sanou Dia - Even with VA supplies, intestinal parasites are a big problem. Lack of footwear and clean water cause them to return, and hinder vitamin absorption.
- Dr. Paul E. Mains - There is merit to producing thymol as worms and intestinal parasites may also develop resistance to anthelmintic drugs.
- Lourlin Ugdimay - Something about nutrition deficiencies and how they are distributed, maybe even purga.
- Dr. John Gileard - Parasites are not only problematic in humans, but also livestock. It is important to address this because people from vulnerable areas rely on their livestock for food and income, and there are only 2 main anthelmintic drugs used in the industry, and snps can easily happen.
After our conversations with our HP contacts, we realized that we had to do something about the problem. Since our yeast is meant to be consumed as a nutritional supplement, we need a compound that is a naturally-occuring anthelmintic that is safe for consumption. [insert transition to thymol].
Thymol is a monoterpenoid found in thyme leaves (Thymus vulgaris). It has long been used in traditional medicine for its antioxidant, anti-inflammatory, and antihyperlipidemic properties (Meeran et al. 2017). Although thymol is “Generally Recognized As Safe” (GRAS) by the United States Food and Drug Administration and may be ingested with “negligible toxicity”, extreme doses may induce unwanted side effects such burning pain in the oesophagus, nausea, abdominal pain, vomiting, and dizziness among others (CITE: SCBT Datasheets). This compound is normally administered through essential oils, tablets, or used as a disinfectant by integration in soaps.
INSERT study about thymol pharmacokinetics. And how oil form is absorbed more quickly because thymol is lipophylic, so yarrowia is a good chassis!
Since Y. lipolytica already has the MEP pathway and produces GPP we will only need the addition of two genes: a terpene synthase (TvTPS1) and a cytochrome P450 (CYP71D178).
PART DESIGN
Thoughtful design of genetic constructs
Thymol parts
EXPERIMENTAL DESIGN
Thoughtful design of experiments
[Insert thymol lab workflow graphic here]
To express thymol in Yarrowia lipolytica a terpene synthase (TvTPS1) and a cytochrome P450 (CYP71D178) will be introduced. To keep substantial DNA stocks we will first transform our constructs in pSB1A3 into chemically competent Escherichia coli DH5α. This DNA would then be extracted through plasmid miniprep. Since Y. lipolytica functions best with non-homologous end joining, we will linearize the plasmid from E. coli with (insert restriction enzyme here) then transform it into Y. lipolytica using lithium acetate transformation which will result in random integration into its genome.
Can still express in E. coli even though it’s codon-optimized for Yarrowia lipolytica.
Ni-NTA purification
Western Blot
Bradford Assay
TvTPS1
To characterize the TvTPS1 gene, just add GPP.
Then check absorbance values ?
CYP71D178
Gamma-Terpinene.
Caenorhabditis elegans is the gold-standard for testing anthelmintic drugs extensively before its clinical trials in humans. To prove thymol’s activity we will be testing its effects on C. elegans by soaking Escherichia coli in varying thymol concentrations, then feed it to the C. elegans. (Observe movement for four days. ). Need to know which concentrations are lethal, how much our
Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.
Expected Results
For when we get back to the lab
In order to provide a sustainable, community-based solution, we plan to genetically modify Rhodosporidium toruloides, an oleaginous yeast that naturally produces beta-carotene and lipids, to be more robust and resource-efficient. By modifying the yeast to produce cellulase, it can then use common agricultural waste products as an energy source for synthesizing its oil. It can then be eaten as a vitamin A supplement. The yeast strain, while naturally safe and non-pathogenic, will also be genetically modified to include a kill switch for bio-containment, and optimized for oil production.
To express thymol in Yarrowia lipolytica a terpene synthase (TvTPS1) and a cytochrome P450 (CYP71D178) will be introduced. To keep substantial DNA stocks we will first transform our constructs in pSB1A3 into chemically competent Escherichia coli DH5α. This DNA would then be extracted through plasmid miniprep. Since Y. lipolytica functions best with non-homologous end joining, we will linearize the plasmid from E. coli with (insert restriction enzyme here) then transform it into Y. lipolytica using lithium acetate transformation which will result in random integration into its genome.
Can still express in E. coli even though it’s codon-optimized for Yarrowia lipolytica.
Ni-NTA purification
Western Blot
Bradford Assay
TvTPS1
To characterize the TvTPS1 gene, just add GPP.
Then check absorbance values ?
CYP71D178
Gamma-Terpinene.
Caenorhabditis elegans is the gold-standard for testing anthelmintic drugs extensively before its clinical trials in humans. To prove thymol’s activity we will be testing its effects on C. elegans by soaking Escherichia coli in varying thymol concentrations, then feed it to the C. elegans. (Observe movement for four days. ). Need to know which concentrations are lethal, how much our
Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.
FUTURE DIRECTIONS
Next Steps
In order to provide a sustainable, community-based solution, we plan to genetically modify Rhodosporidium toruloides, an oleaginous yeast that naturally produces beta-carotene and lipids, to be more robust and resource-efficient. By modifying the yeast to produce cellulase, it can then use common agricultural waste products as an energy source for synthesizing its oil. It can then be eaten as a vitamin A supplement. The yeast strain, while naturally safe and non-pathogenic, will also be genetically modified to include a kill switch for bio-containment, and optimized for oil production.
References
Paper 1
