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− | + | Cloning | |
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− | + | 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. | |
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− | + | CONFIRMATION OF THYMOL PRODUCTION/ ENZYME CHARACTERIZATION | |
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− | + | 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. | ||
+ | |||
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− | + | CAENORHABDITIS ELEGANS TESTING | |
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− | + | 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 | |
+ | |||
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Revision as of 02:03, 9 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.
![](https://static.igem.org/mediawiki/2020/9/99/T--Calgary--thymoloverview.jpeg)
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.
UNDERSTANDING THE PROBLEM
Who we talked to
To understand the extent of the problem, we talked to people who work directly with the problem.
![](https://static.igem.org/mediawiki/2020/4/47/T--Calgary--ThymolHP.jpeg)
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.
PART DESIGN
Thoughtful design of genetic constructs
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.
EXPERIMENTAL DESIGN
Thoughtful design of experiments
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.
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.