Team:Calgary/Biocontainment Engineering
Overview
Understanding the Problem
Research and Ideate Solutions
Part Design
Experimental Design
Results
Future Directions
References
OVERVIEW
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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:
- Understand the Problem
- Research and Ideate Solutions: Identifying a biocontainment strategy
- Design Solutions: Part design
- Design Solutions: Experimental design
- Create: Results and Troubleshooting
It is important to note that there will be circular action in this process, particularly between steps 4-5 (i.e the results of step 5 informing iterations of step 4). However, we are currently presenting one turn of the engineering design cycle by working towards a lab proof of concept. In reality, all of these steps will repeat once we have accomplished our goals/tested our product in the lab and look towards community implementation. We highlight our plans beyond iGEM in the Future Directions section.
UNDERSTANDING THE PROBLEM
Why is biocontainment important?
Synthetic biology is an emerging field that promises scalable and low-cost solutions to global health, agricultural, and environmental problems in the form of self-replicating biological devices. As these solutions increasingly become a reality, they migrate from tightly regulated laboratories to private spaces, such as households. This raises significant safety concerns regarding biocontainment, as the introduction of non-native organisms into the environment can result in unintended adverse effects on the ecosystem. Oviita is a community product that is meant to be cultivated outside of the laboratory by the people in the community, as such ensuring high levels of biosafety was a crucial part of our project. In order to ensure safety, iGEM Calgary set out to design a robust, yet sustainable biocontainment system.
Previous strategies
When we began our journey towards finding an effective biocontainment strategy, we considered many different options including auxotrophy and a kill switch system, before eventually settling on our final strategy of a complementary auxotrophy co-culture system.
Auxotrophy prevents the production of a vital molecule by an organism, thereby forcing the organism to become reliant on the external supplementation of the molecule of interest for survival (ND1). A positive side to auxotrophy is the low mutation rate of reverting the auxotrophy back to prototrophy. Find reference that says this. However, such a system introduces high costs, and is ultimately unsustainable because of the need for supplementation.
An alternative biocontainment strategy is the introduction of a kill switch system. Such a system comes with its own positive aspects and limitations. Kill switch systems are genetic circuits that lead to the production of a toxin or inhibitory molecule following exposure to a certain stimulus, resulting in the death of the organism (1). For example, a kill switch system could involve the production of a nuclease in response to blue light. However, some limitations for this method is that the system relies upon the availability of the stimulus in order to be effective. In addition, when we talked to Dr. Robert Mayall, he explained how there is a higher rate of mutating out the kill switch system due to the fitness cost instilled in an organism.
Bioreactor Engineering Controls
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.
RESEARCH AND IDEATE SOLUTIONS
Initial ideas
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.
Finalized strategy
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
Genetic Constructs
In order to implement our biocontainment strategy in Y. lipolytica , we need to engineer two strains of yeast; one that is auxotrophic for tryptophan and overproduces leucine, and one that is auxotrophic for leucine and overproduces tryptophan.
Engineering amino acid overproduction
EXPERIMENTAL DESIGN
Proof of concept
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.
Auxotrophy
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Amino acid overproduction
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.
RESULTS
Auxotrophy
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.
Amino acid overproduction
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
Improving our system
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.
Engineering Yarrowia lipolytica
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
- ND1: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264511/
