Difference between revisions of "Team:Calgary/Thymol Engineering"

Line 186: Line 186:
 
         <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.
+
To allow our target communities to maximize micronutrient and vitamin A absorption in between deworming seasons, we will engineer our yeast to produce thymol which is naturally found in thyme leaves. This will allow our yeast consumers to alleviate damage and destroy parasitic worms in the intestines. Our initial goal is to create a proof of concept in the lab. To do this, we followed the engineering design cycle to build our project:
 +
 
 +
 
 +
 
  
 
         </p>
 
         </p>
 +
 +
<p> Over the next year, we will be working towards a lab proof of concept for thymol testing and production. This engineering design cycle will repeat once we have accomplished this and look towards community implementation. For more information on our plans for next year and beyond iGEM, please refer to the Future Directions section.</p>
 +
 
<center><img style="width:60%;"src="https://static.igem.org/mediawiki/2020/d/d3/T--Calgary--Thymol_and_parasite.png"></center>
 
<center><img style="width:60%;"src="https://static.igem.org/mediawiki/2020/d/d3/T--Calgary--Thymol_and_parasite.png"></center>
 
<p style="color:grey; font-size: 85%;"> Figure 1. Intestinal parasites (left) and the molecular structure of thymol (right). </p>
 
<p style="color:grey; font-size: 85%;"> Figure 1. Intestinal parasites (left) and the molecular structure of thymol (right). </p>
Line 202: Line 208:
 
<div class = "understanding-the-problem" id="understanding-the-problem">
 
<div class = "understanding-the-problem" id="understanding-the-problem">
 
         <h2>UNDERSTANDING THE PROBLEM</h2>
 
         <h2>UNDERSTANDING THE PROBLEM</h2>
−
         <h4>Who we talked to</h4>
+
         <h4>Inadequate Access to Healthcare</h4>
 
         <p>  
 
         <p>  
−
        To understand the extent of the problem, we talked to people who work directly with the problem.  
+
        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, 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 using agents like albendazole and mebendazole, but lack of clean water and proper footwear allow these parasites to return and thrive in the intestines. </p>
−
        </p>
+
  
 +
<p>Parasitic worms are among the most widespread human infections to date, affecting 2 billion people worldwide. The most common of these are Ascaris lumbricoides or the giant roundworm, hookworms, and Trichuris trichiura or the human whipworm. These worms may be obtained from direct contact with the soil, or through improper measures in hygiene. These worms cause a variety of symptoms such as stunted growth, decreased metabolic rates, physical and mental fatigue, and malnutrition (Kumar, Jain, & Jain, 2014). Since these parasites perforate the intestines, nutrition absorption is severely inhibited, causing several micronutrient supplementation programs to falter. </p>
  
−
        <img class="img-fluid"src="https://static.igem.org/mediawiki/2020/4/47/T--Calgary--ThymolHP.jpeg">
+
<p>Albendazole has been used as an effective cure for intestinal worm infections. When treating roundworms it has a cure rate of 98.9% and egg reduction rates of 99.6%. For hookworms, it is 56.8% and 97.7% respectively, while in whipworms it is 10.% and 73.3%. However, even with these highly effective drugs the worms return and thrive until the next round of deworming because of poor hygiene due to lack of access to clean water and proper footwear. </p>
−
<p style="color:grey; font-size: 85%;"> Figure 1. Dr. Sanou Dia, Dr. Paul E. Mains, Lourlin Ugdimay, and Dr. John Gileard (from left to right). </p>
+
  
−
        <p>
+
<p>Talking to Dr. John Gilleard and Dr. Paul E. Mains from the University of Calgary, we found out that these drugs were first intended for animals but were later implemented in humans. They believe that intestinal parasites will sooner or later develop drug resistances just as they have discovered in sheep and cattle models, which is why development of new treatments should be kept on the radar. </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>
+
  
−
<p>
+
        </p>
−
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].</p>
+
  
−
<p>
 
−
<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. </p>
 
  
−
<p>
 
−
INSERT study about thymol pharmacokinetics. And how oil form is absorbed more quickly because thymol is lipophylic, so <span style="font-style:italic">Y. lipolytica</span> is a good chassis! </p>
 
  
−
<p>
 
−
Since <span style="font-style:italic">Y. lipolytica</span> 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>
 
−
       
 
−
        </p>
 
 
</div>
 
</div>
 
<br>
 
<br>
Line 240: Line 229:
 
<hr>
 
<hr>
 
<div class = "part-design" id="part-design">
 
<div class = "part-design" id="part-design">
−
         <h2>PART DESIGN</h2>
+
         <h2>IDENTIFYING THYMOL</h2>
−
         <h4>Thoughtful design of genetic constructs</h4>
+
         <h4>Why produce an anthelmintic?</h4>
 
         <p>
 
         <p>
−
           Thymol parts
+
           For this part of the project, we initially intended to produce omega-3 fatty acids such as eicosapentaenoic acid (EPA) which has cognitive benefits to improve the nutritional value of our yeast. We learned from Dr. Dia Sanou and Lourlin Ugdiman, two public health experts who have worked in Africa and the Philippines, that producing a safe and supplementary anthelmintic as a temporary measure is a better use of our efforts. That temporary measure would be sufficient until the next round of deworming. </p>
 +
 
 +
<p>Our team started looking for plant-based compounds with promising effects for anthelmintics against nematodes. We came across thymol, a compound which is the primary component in thyme oil. Thymol has been used with epsom salts to cure hookworm infections in rural schoolchildren as early as 1912. Half a gram would be administered to children ages 1-5 and a gram for those aged 5-10 (Rural School and Hookworm Disease p. 12, 1970). In modern times, the compound can be found in products such as soaps, disinfectants to pesticides, and in anti-inflammatories.  It is labeled to be safe for ingestion with “negligible toxicity” by the USFDA in humans. However, in high doses it may cause burning pain in the oesophagus, nausea, abdominal pain, vomiting, and dizziness [CITE DATASHEET].
 
         </p>
 
         </p>
 
</div>
 
</div>

Revision as of 00:28, 27 October 2020



OVERVIEW




Optimizing Nutrient Absorption Through Anthelmintics

To allow our target communities to maximize micronutrient and vitamin A absorption in between deworming seasons, we will engineer our yeast to produce thymol which is naturally found in thyme leaves. This will allow our yeast consumers to alleviate damage and destroy parasitic worms in the intestines. Our initial goal is to create a proof of concept in the lab. To do this, we followed the engineering design cycle to build our project:

Over the next year, we will be working towards a lab proof of concept for thymol testing and production. This engineering design cycle will repeat once we have accomplished this and look towards community implementation. For more information on our plans for next year and beyond iGEM, please refer to the Future Directions section.

Figure 1. Intestinal parasites (left) and the molecular structure of thymol (right).



UNDERSTANDING THE PROBLEM

Inadequate Access to Healthcare

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, 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 using agents like albendazole and mebendazole, but lack of clean water and proper footwear allow these parasites to return and thrive in the intestines.

Parasitic worms are among the most widespread human infections to date, affecting 2 billion people worldwide. The most common of these are Ascaris lumbricoides or the giant roundworm, hookworms, and Trichuris trichiura or the human whipworm. These worms may be obtained from direct contact with the soil, or through improper measures in hygiene. These worms cause a variety of symptoms such as stunted growth, decreased metabolic rates, physical and mental fatigue, and malnutrition (Kumar, Jain, & Jain, 2014). Since these parasites perforate the intestines, nutrition absorption is severely inhibited, causing several micronutrient supplementation programs to falter.

Albendazole has been used as an effective cure for intestinal worm infections. When treating roundworms it has a cure rate of 98.9% and egg reduction rates of 99.6%. For hookworms, it is 56.8% and 97.7% respectively, while in whipworms it is 10.% and 73.3%. However, even with these highly effective drugs the worms return and thrive until the next round of deworming because of poor hygiene due to lack of access to clean water and proper footwear.

Talking to Dr. John Gilleard and Dr. Paul E. Mains from the University of Calgary, we found out that these drugs were first intended for animals but were later implemented in humans. They believe that intestinal parasites will sooner or later develop drug resistances just as they have discovered in sheep and cattle models, which is why development of new treatments should be kept on the radar.



IDENTIFYING THYMOL

Why produce an anthelmintic?

For this part of the project, we initially intended to produce omega-3 fatty acids such as eicosapentaenoic acid (EPA) which has cognitive benefits to improve the nutritional value of our yeast. We learned from Dr. Dia Sanou and Lourlin Ugdiman, two public health experts who have worked in Africa and the Philippines, that producing a safe and supplementary anthelmintic as a temporary measure is a better use of our efforts. That temporary measure would be sufficient until the next round of deworming.

Our team started looking for plant-based compounds with promising effects for anthelmintics against nematodes. We came across thymol, a compound which is the primary component in thyme oil. Thymol has been used with epsom salts to cure hookworm infections in rural schoolchildren as early as 1912. Half a gram would be administered to children ages 1-5 and a gram for those aged 5-10 (Rural School and Hookworm Disease p. 12, 1970). In modern times, the compound can be found in products such as soaps, disinfectants to pesticides, and in anti-inflammatories. It is labeled to be safe for ingestion with “negligible toxicity” by the USFDA in humans. However, in high doses it may cause burning pain in the oesophagus, nausea, abdominal pain, vomiting, and dizziness [CITE DATASHEET].


EXPERIMENTAL DESIGN

Thoughtful design of experiments

Add a blurb

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