Team:Toronto/Demonstrate

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Demonstrate

In many practical industrial contexts, large volumes of water must be processed employing phase separation techniques, and these make use of largely mechanical techniques (e.g. centrifugation) which are energy inefficient and costly. Even alternative methods like reverse osmosis require pressurization, which can require a great deal of energy for large volumes. Our Policy and Practices Team established the need for a renewable, energy efficient bioremediation technique within the industries of mining operations and wastewater treatment plants. Developing a biological platform for solid phase extraction was the main goal of our project.

In this project, we explored the application of gas vesicles as a synthetic biology tool for bioremediation. Inspired by promising results from Bordeau et al., who noticed cellular flotation when investigating a synthetic gas vesicle construct for ultrasound imaging. Previous iGEM teams’ experimental results from genetic modification of E. coli cells to express gas vesicles and perform floatation has been inconsistent and inconclusive. To our knowledge there has been no scientific research or experimentation, outside of iGEM, that optimises the protocol for cellular floatation in context of bioremediation. Additionally, functions of gas vesicle operons and their proteins has not been adequately characterised. What is known, however, is that GvpA and GvpC are essential for floatation; GvpA being a being the primary structural protein and GvpC being a scaffold protein providing stabilization to the gas-filled proteinous structures. We acquired qualitative observations and quantitative OD readings and dry mass of cells at different depths per unit of time. We found that our pET28a construct containing Arg1 performed floatation in LB media over a 25 hour period after induction by IPTG, unlike the negative control. Furthermore, we found that the quality of floatation was sensitive to the OD at which the cells were induced.

Our team aimed to create an optimized system that could be applicable to multiple waste water processing contexts involving bioremediation. This is particularly emulated by our dry lab’s bioreactor model. This model is modular in that it has many adjustable parameters that can be set based on empirical values pertaining to different bioremediation tasks (e.g. the rate constant for binding of particle to engineered cell surface receptors), allowing for prediction of efficiency and performance for many different bioremediation tasks.

Based on the results of our differential bioreactor model, we postulate that a bioreactor of this design could perform at appropriately small time-scales with a sufficiently optimized flotation construct in a bioremediation context. This is useful for model validation, and proof of concept. This bodes well for future laboratory endeavours where the bioreactor schema along with an engineered cell-line optimized for flotation from gas vesicle formation could be tested in a small scale laboratory model of the system to test its empirical performance. For future application-based analysis using the bioreactor model, Goempertz coefficients could be determined for a biomass of industrially relevant size for the volume demands dictated by the industry requiring bioremediation using a similar experimentation and analysis technique as described in the Growth Dynamics section (link to models page). We also designed a stochastic temporal tracking algorithm to acquire real flotation data (images) to estimate the buoyant force for ARG1 and compare different modifications of ARG1 to determine an optimal gene combination for flotation. To further enrich the analysis of the behaviour of the biomass in the bioreactor model, in addition to the existing sensitivity analysis, identification and characterization of stable points resulting from different combinations of parameter values (like maximum carrying capacity of biomass) could be performed to model a system where biomass performs functions like binding to organic or inorganic molecules of interest, before floatation based separation.



The theoretical framework built by our team can be used in the future to test and produce an effective and efficient bioremediation method that far surpasses conventional methods in use today. This innovative approach to facing waste water provides the world with a more independence and variety in the compounds that can be extracted. As global water security is becoming a pressing concern, Our project is relevant to both issues facing our local communities and the entire world as a whole.