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

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Revision as of 07:21, 25 October 2020



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.

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



UNDERSTANDING THE PROBLEM

Who we talked to

To understand the extent of the problem, we talked to people who work directly with the problem.

Figure 1. Dr. Sanou Dia, Dr. Paul E. Mains, Lourlin Ugdimay, and Dr. John Gileard (from left to right).

  • 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