Team:Calgary/Engineering
OVERVIEW
The Engineering Design Cycle
To address each component of the Oviita pipeline, we followed the engineering design cycle to build all of our solutions:
- Understand the Problem
- Research and Ideate Solutions
- Design Solutions
- Create: Expected results and troubleshooting
- Testing
- Repeat
While each of our subprojects are at different points and iterations of this cycle, this general workflow was adapted to every component of Oviita. This page provides an overview of the engineering success for all of our subprojects, but please visit the subproject-specific pages to learn more!
Featured Part Characterization
This year was a unique iGEM season. In a typical year, the pinnacle of engineering success in iGEM projects in seen in the creation and characterization of parts. While we dedicated a lot of time to creating parts this year (learn more on our parts or part collection pages), we were unable to characterize them in the lab due to COVID-19 restrictions. However, two of our parts in particular: BBa_K3629016 and BBa_K3629013 were unique in having extensive modelling and specific design considerations involved in their creation. These features are explained below to characterize these parts without lab data yet. We also briefly explain our plans to characterize these parts in the lab, but you can learn more about these plans on our characterization page.
Modified TrEGI: BBa_K3629016
EGI or endoglucanase I is an essential enzyme required for cellulase activity that functions by cleaving internal beta-1-4 bonds in cellulose polymers.
MODELLING
For the modified and wild type enzyme, we predicted the three-dimensional structure using homology modelling. From these structures, we completed molecular dynamic simulations. These simulations were then characterized by GausHaus to determine if the changes increased variance in the enzyme dynamics. The modifications actually had a net lowering of the variance in the enzyme allowing the team to move forward confident in the modifications. Learn more on our modelling page.
Modified PfCBHI: BBa_K3629013
CBHI or cellobiohydrolase I is another essential enzyme required for cellulase activity that functions by freeing cellobiose units from broken cellulose polymers produced by endoglucanses. CBHI specifically does this on the reducing end of the sugar molecule.
MODELLING
Structural models were generated to get a starting point for the protein. The predicted structure was then protonated in silico at numerous pHs. After protonation, the structures were solvated in water and underwent molecular dynamic simulation. Metrics around the simulations were taken and modelled to show protein stability within the pH 3 to 7 range. Learn more our modelling page.
WET LAB
Cellulase Engineering
Go to Cellulase Engineering page
Cellulase integration is an essential part of Oviita. Successful integration of the cellulase genes make Y. lipolytica capable of growing on inexpensive and readily available cellulose material. This allows our yeast to be easily cultivated by VAD communities. With plans to build Oviita over the next two years, it is important we follow sequential steps to engineering design to ensure success in this subproject.
We first consulted academic experts, Dr.Raymond Turner, PhD student Trevor Randall and Dr. Vanina Zaremberg, on the design of our project and the best cloning techniques to use to accomplish our goals. Once we decided on Gibson Assembly, we moved forward with picking our cellulases and optimizing their function for Y. lipolytica. From extensive literature review, we were able to design our constructs based on previous successful studies, and submit these parts to the registry. We have since made extensive experimental plans to engineer these parts into Y. lipolytica and characterize our cellulase parts individually and as functional proteins in a co-culture system. This design stage is important, but also subject to revision as we get into the lab and experience unexpected results or have failed experiments. However, we were able to test the general idea of Y. lipolytica cultivation on cellulose material (supplemented with cellulase) through our preliminary cellulase experiments. The results from these experiments will inform our genetic engineering experiments to ensure enough cellulase is being produced by the engineered strains.
Biocontainment
Go to Biocontainment page
Through the course of the season, we progressed through different iterations of the engineering design cycle. First, by conducting extensive literature review and speaking to experienced members in academia, we explored different ideas for our biocontainment system such as a light-induced kill switch system, nucleases, and anti-toxin system. These different strategies each went through separate iterations of the design cycle, which then led us to eventually settle on our finalized biocontainment strategy: a complementary co-culturing system, wherein one strain will overproduce an amino acid that the second strain is auxotrophic for, making the two strains dependent upon each other for survival, and thus unable to proliferate outside of the intended growth vessel. We then progressed to the next stage of the cycle, where we designed experiments to test our biocontainment system. Specifically for this season, we decided to focus on developing a proof of concept using a pair of already engineered S. cerevisiae strains. By characterising these strains in terms of auxotrophy and overproduction of amino acid, developing a co-culture of the strains, and testing their growth in environmental conditions, we collected preliminary data that can be used to inform the next stage of our project: the engineering of Y. lipolytica strains to create a biocontainment system to be implemented for the purposes of this project. You can learn more about how we followed the design cycle and what we learned through the process here.
DRY LAB
Bioreactor
Go to Bioreactor page
When people hear the word bioreactor, they think something of some complicated concept found in science fiction or advanced industry. We have made an effort to make it the exact opposite. Our intention has been to develop the Field Adaptable Bioreactor (FAB) around simplicity, sustainability, and flexibility. We believe we can accomplish this with the help of our Lab Adaptable Bioreactor (LAB). Due to the adaptable design of the FAB, we are essentially giving our users the creative freedom to use whatever materials they may have at hand; encouraging the act of recycling scrap materials and increasing accessibility.
Randle's Cell Testing Device
Go to Randle Cell Testing Device page
We learned from Dr. Charles Mather that there was difficulty obtaining data on Vitamin A, and further consulted Banda Ndiaye, who indicated that decision makers needed more data for intervention. We also learned from Dr. Sanou Dia, that a portion of data on VAD is based on coverage and not diagnosis. Inspired by low-cost, electrochemical detection circuits, the Randle Cell Circuit was born. We investigated what biomarker we could test to identify VAD, and consulted Dr. Christopher Naugler to learn about current methods. After identifying RBP as an indicator of VAD status, we set to work laying framework for a diagnostic test. Given the scope of this task, and lack of lab access, we decide to focus on the hardware needed to quantify retinol binding protein in a blood sample. We consulted experts such as Colin Dalton, Sultan Khetani, and Thomas Lijnse for guidance on what biosensor system to select, as well as techniques to analyze them. Ultimately, we were able to source and program hardware capable of measuring impedance of capacitive loads in a step towards analyzing blood samples. We conducted simple tests, running frequency sweeps on loads of varying resistance and capacitance. Once we confirmed that these measurements were accurate, with a percent error as low as 0.02%, we tested this device in an aqueous solution. As a proof of concept, this indicated that the hardware is capable of quantifying impedance in an electrochemical cell.
