Engineering

Introduction Genetic Circuit Implementation Enzyme choice Model Conclusion Future Perspective

Introduction

The development of Mhetyguá’s project was meticulously grounded in solid engineering practices to optimize both the progression of the project and the excellence of the end product. Throughout the project, we employed several iterative engineering cycles, encompassing design, build, test, and learn phases, to establish a dependable system. Leveraging these principles, we effectively constructed our project for microplastic degradation. A more detailed breakdown of these guiding principles and steps can be found on the subsequent page, with a special emphasis on its application in Chlamydomonas Reinhardtii. We also talked to stakeholders and researchers to gain knowledge we otherwise would not have obtained. These warranted repeated changes to our design and tweaks to aspects of our project.

Genetic Circuit

We recommend reading the design page first to understand the context of our genetic circuit design and how the DBTL cycle was applied.

Cycle 1

  • Design: The initial design of our circuit was intricate and expansive, employing the Golden Gate assembly methodology. This design was informed by the research conducted by prior iGEM teams and pertinent scientific literature in the domain.
  • Build: Utilizing Benchling, we engineered the primary circuit, incorporating essential modifications to ensure effective expression in both Chlamydomonas reinhardtii and Escherichia coli.
  • Test: The assembly was simulated using Benchling to evaluate its feasibility.
  • Learn: Post in-silico evaluations indicated that the assembly process would be protracted, given the multitude of assemblies planned and the anticipated delivery timeline of our syntheses. Consultations with our advisors, João Molino, Samuel Chagas, Matheus Araujo, Giulio Bratz, and our Principal investigator, Cristian Rojas, led to the consensus that a more streamlined circuit, retaining the capability to express our target enzyme, would be more pragmatic.

Cycle 2

  • Design: The conceptualization for our secondary circuit was influenced by prior research undertaken by our advisor, João Molino. He advocated for a more simplified circuit design. Concurrently, our lead researcher, Cristian Rojas, recommended the adoption of the Gibson Assembly methodology for our assemblies, given the pre-existing laboratory resources compatible with this technique, eliminating the need for additional acquisitions of reagents.
  • Build: The circuits were meticulously designed in Benchling, tailored for both Chlamydomonas reinhardtii and Escherichia coli. Presently, we are in the advanced stages of assembling our plasmids embedded with the circuit.
  • Test: To be conducted in the imminent future.
  • Learn: A salient insight we have gleaned thus far is the occasional necessity for minimalistic approaches, especially when constrained by time. Such an approach ensures the optimization of our scientific endeavors.

Implementation

Cycle 1

  • Design: At the beginning of our research, we identified the possibilities where the project could be implemented, considering our chassis, releasing it into a body of water like a river was not an option, due to biosecurity concerns. So we decided that our implementation would be inside a photobioreactor.
  • Build: Since we don't have a photobioreactor in our laboratory, we would have to buy or build one. The option to buy was not viable due to economic constraints. So we decided to build a photobioreactor, for which we contacted Victor Marchesan, who developed the Open-Source Modular Bioreactor, hardware from the 2022 iGEM UFMG_UFV team.
  • Test: Following discussions with individuals experienced in photobioreactor design, we formulated a theoretical prototype and made a budget for purchasing the parts.
  • Learn: We realized that building a photobioreactor was not a priority, as we would have to cover the costs of the entire competition, even though the budget was not large, we needed to save the money. Furthermore, the need to advance our research to innovate in the development of a homemade PBR became evident, given that certain iGEM teams had already successfully created such hardware. Given these constraints, both resources and time were not in our favor.

Cycle 2

  • Design: After seeing the infeasibility of building our photobioreactor, we explored alternative methods for facilitating enzymatic degradation. As a result, we considered the possibility of enzyme immobilization.
  • Build: We conducted research on enzyme immobilization techniques and determined that applying enzymes within alginate beads, a material frequently employed for this purpose, was the most suitable approach. The ultimate implementation of this method would be within water treatment plants.
  • Test: Upon reaching out to the local sanitation institution, we were informed that employing biological techniques for water treatment might not be the most suitable approach, as they primarily rely on physical-chemical methods. Consequently, they recommended the use of immobilized enzymes for sewage treatment, where biological methods were already in practice.
  • Learn: Following discussions with stakeholders, we also conducted a comprehensive economic analysis and determined that a more cost-effective approach would be to directly apply the microalgae to the sewage treatment plant. This would allow the microalgae to secrete the enzymes naturally, eliminating the need for enzyme isolation, which tends to increase process costs.

Cycle 3

  • Design: Finally, we directed our research towards a more specific and contextual area. It became evident that implementing our solution in wastewater treatment plants was the most practical choice. We identified stabilization ponds as the optimal location for our project implementation, given that these ponds naturally support the growth of microorganisms like microalgae.
  • Build: We focused on understanding the operational dynamics of stabilization ponds, in addition to seeking data on the socio-environmental context and the utilization of this sewage treatment approach in Brazil.
  • Test: To conduct a proof of concept, we cultivated our microalgae in TAP culture medium with samples of wastewater at different concentrations (10%, 25%, 50%, 75%, and 100%).
  • Learn: We are currently in the phase of analyzing the growth rate in these conditions to later compare it with the widely used TAP culture medium and define new actions for the project.

Enzyme choice

Cycle 1

  • Design: In the early stages of our research, we embarked on a quest to identify the most suitable enzyme for PET degradation. We encountered several options, including PETase, LCC, and PHL7. To make an informed choice, we conducted thorough comparisons among them (check our Design page here), meticulously evaluating the respective advantages and disadvantages associated with each enzyme.
  • Build: We finally opted for PHL7 due to its remarkable stability and superior PET degradation rate, especially at elevated temperatures like 70°C. This decision was aligned with our initial implementation concept, which involved a bioreactor setup. Consequently, we proceeded to construct our circuit incorporating PHL7.
  • Test: Since we didn't have any plasmid that expressed PHL7, we were looking for a suitable one. Fortunately, our advisor, João Molino, generously donated one to our project. This proved to be immensely beneficial, saving us from the 45-day waiting period typically associated with obtaining a synthesis from IDT.
  • Learn: Throughout the progression of our project, we underwent several iterations in our implementation strategy. These adjustments significantly influenced our selection of the enzyme of interest. Notably, as we shifted our focus to the utilization of a stabilization pond, it became evident that PHL7 would no longer operate at its peak catalytic efficiency since the temperature within the stabilization pond is lower than the enzyme's optimal 70°C operating range.

Cycle 2

  • Design: Considering the final implementation idea (check our implementation here), we explored alternative enzymes and came across FAST-PETase, an enzyme notable for its commendable catalytic rate in PET degradation, particularly well-suited for the temperature typically found in stabilization ponds.
  • Build: Considering the new enzyme, FAST-PETase, we proceeded to redesign our circuit, optimizing it for Chlamydomonas reinhardtii and Escherichia Coli, then subsequently we built our plasmid based on the previous construction. Essentially, our design only entailed replacing PHL7 with FAST-PETase.
  • Test: We are in the phase of building our plasmid, first linearizing our plasmids using PCR and then assembling them with Gibson Assembly's FAST-PETase.
  • Learn: After building our plasmid we will move on to the analysis phase of the catalytic activity and the expression rate of this enzyme to then compare it with PHL7 and define new actions for the project.

Model

Cycle 1

  • Design: Our first modeling idea came from the concept of ecological modeling, which incorporates a variety of ecological factors such as species interactions, energy flow, nutrient cycles, population dynamics, and climate change.
  • Build: The aim was to determine the ultimate biomass of our microalgae by constructing a model that delineates interactions between species.
  • Test: While the concept of ecological modeling intrigued us, we found it unsuitable for the initial phase of the project. This decision was influenced by biosafety and implementation concerns, as well as the fact that it was an entirely new area for our team members, this posed potential challenges given our limited time frame. Furthermore, we lacked a network of individuals experienced in this specific modeling field who could provide guidance and support.
  • Learn: We gained familiarity with fundamental ecological modeling concepts that could be integrated into other modeling methods, which we had previously had contact with.

Cycle 2

  • Design: With the implementation of the project aimed at stabilization ponds, we had the idea of modeling something that involved this topic, especially because other iGEM teams had already worked with PET degrading enzymes in microalgae, so we would like to design our unique model. So, we decided to kinetically model the simultaneous growth of our microalgae and the PET degradation in sewage, while also structurally modeling an enzyme-linker complex.
  • Build: Overall, a lot of literature research was necessary, as well as inspiration from models similar to the context of our project. To develop the kinetic and structural analyses, we used computational resources, in addition to previously described data and equations.
  • Test: As we carried out our simulations, some adaptations were necessary so that we could reach more satisfactory and coherent results. For example, the first analyzes of the graphs completely changed the integration structure of our model, moving from differential equations to the Gompertz model. If you want to understand the modeling process and more adaptations done, check our Model page here.
  • Learn: In the example mentioned above, we understood that our modifications to the base model we used directly influenced the final analysis, which made us insert a growth model based on the sigmoidal microalgae growth curve to generate truly meaningful comparisons.

Conclusion

The Mhetygua project showcases a systematic and iterative approach to addressing the challenge of microplastic degradation. By employing sound engineering practices, the team navigated through various phases of design, build, test, and learn, ensuring that each step was grounded in research, expert advice, and practical considerations. The project's adaptability was evident in the shifts in implementation strategies, from photobioreactors to wastewater treatment plants, and in the enzyme choices, transitioning from PHL7 to FAST-PETase based on efficiency and environmental conditions.

Future Perspective

Moving forward, the Mhetygua project holds significant promise in the realm of environmental biotechnology. As the team continues to analyze the catalytic activity and expression rate of FAST-PETase, there is potential for further optimization and scalability. Collaborations with wastewater treatment facilities could pave the way for real-world applications, transforming the way we address microplastic pollution. Additionally, the project serves as a blueprint for future iGEM teams and researchers, emphasizing the importance of adaptability, stakeholder engagement, and iterative design in scientific endeavors. As the global community becomes increasingly aware of environmental challenges, projects like Mhetygua will be at the forefront of sustainable solutions.