CONTRIBUTION
Facing epilepsy, a long-standing and difficult-to-treat disease worldwide, the ketogenic diet therapy has long been regarded as an effective adjunctive treatment, showing remarkable efficacy in suppressing epileptic seizures. In the ketogenic diet, β-hydroxybutyrate (BHB) is a crucial functional molecule, which can treat epilepsy through mechanisms such as increasing gamma-aminobutyric acid (GABA) in the brain and the GABA/glutamate ratio. The development of this therapy is of long-term significance and necessity from a therapeutic perspective. Our team has focused on the pain points in the ketogenic diet process—such as its side effects and low adherence. We have always maintained close contact with stakeholders, and proposed a project pursuit to transplant the ketogenic process into engineered bacteria and excrete BHB into the human circulation, thereby serving as a substitute for the ketogenic diet.
We attempted to explore and establish a synthetic pathway for the normal synthesis of BHB in the Escherichia coli prokaryotic expression system. This approach has significant advantages over the existing chemical methods for BHB synthesis:
- BHB biosynthesis using glucose as the substrate, with a wide range of substrate sources;
- The production process is completed solely by engineered bacteria, featuring environmental friendliness;
- E. coli can adhere and colonize in the intestinal tract, which helps us improve the drug delivery method and directly excrete BHB for human absorption.
The design of this synthetic pathway was initially inspired by a similar industrial BHB synthesis technology route. However, to enable the successful excretion of BHB from the bacteria, we added the rplO gene, which encodes a BHB transport protein from E. coli, to this route. This laid the foundation for our envisioned in vivo drug delivery. During the experiment, we gained a deeper understanding of this expression process, hoping to provide some ideas for friends in the iGEM community who are also working on the expression of eukaryotic proteins in the prokaryotic expression system:
- Theoretically, bacteria can accommodate multiple plasmids, but there are many influencing factors for their stable existence. We even attempted to use four plasmids in the experimental design stage, but this obviously put a huge pressure on the bacteria. Later, we switched to two plasmids, pRSFDuet-1 and pET-Duet-1, which enabled the successful expression of the target gene. We suggest that when introducing multiple plasmids into a single bacterium, try to choose plasmids from different incompatibility groups to avoid competition for the same replication mechanism and plasmid loss; at the same time, multiple selection pressures can promote the stable coexistence of plasmids from different incompatibility groups, which is related to environmental screening; when conditions permit, prefer smaller plasmids to reduce the metabolic pressure on bacteria and better achieve plasmid coexistence;
- When using the prokaryotic expression system to express prokaryotic proteins, the formation of inclusion bodies is a natural consideration. We effectively reduced the formation of inclusion bodies by seamless cloning and connecting the SUMO tag, generating a large amount of soluble protein. This undoubtedly provided convenience and support for the construction of our synthetic pathway and subsequent protein purification verification.
The optimization of the stability and reliability of this pathway requires a large amount of future experiments and data feedback to achieve. At the same time, the development of protein expression and purification work cannot be separated from the efforts and contributions of every researcher. We hope to call on more people to pay attention to this field through the iGEM platform and join us in researching more efficient, green, and stable BHB synthesis methods!
We further enriched and supplemented the iGEM Registry of Standard Biological Parts. The BHB synthesis pathway built under the prokaryotic expression system requires coding phaA, phaB, pcT and rplO to achieve the synthesis and excretion of BHB in Escherichia coli Nissle 1917. In fact, through our experimental verification, each part of this synthesis pathway functions as expected. Under the detection of the microplate reader, the single colonies of the engineered bacteria finally obtained all showed good BHB product synthesis amounts. This is also a successful engineering cycle.
To ensure that the engineered bacteria are colonized in the small intestine of the digestive tract as per the pre-determined plan, while avoiding complex interactions between the engineered bacteria and the intestinal flora and the resulting genetic material exchange and serious gene leakage and other biosafety issues, we have creatively proposed an experimental design of encapsulating the engineered bacteria in hydrogels. Using multi-layer hydrogels as the delivery system instead of modifying the engineered bacteria to have the ability to colonize has significant advantages:
- Protecting the engineered bacteria from extreme pH environments and ensuring their successful survival, such as in the highly acidic gastric juice of the digestive tract where engineered bacteria find it difficult to survive;
- Physically isolating the engineered bacteria from the intestinal flora, effectively avoiding the aforementioned biosafety issues;
- Introducing pH-responsive coating materials, allowing the hydrogel to remain stable in acidic environments and adhere in the alkaline environment of the small intestine, ingeniously solving the problem of engineered bacteria colonization.
In terms of the specific design, the core of the hydrogel is made of alginate, a biocompatible material that solidifies into spherical droplets when immersed in sterilized CaCl2 solution; the viscous middle layer of the hydrogel is mainly composed of sodium alginate, dopamine hydrochloride, and acrylamide, forming a middle layer coating under the action of crosslinking agents, initiators, and catalysts; the pH-responsive outer layer of the hydrogel is made of calcium alginate gel film formed by the reaction of calcium ions and SA, and it is recommended to be stored in a moist environment.
Designing such a hydrogel-encapsulated engineered bacteria delivery system is highly instructive and valuable in application. More importantly, we have taken an important and imaginative step in the solution to the problem of live bacteria drug delivery. In the Conference of China iGEMer Community (CCiC), we noticed that many teams' projects involved the colonization of engineered bacteria in the human body, but they were somewhat confused about the design in terms of safety and colonization efficiency. Our hydrogel delivery system provides a valuable experience and thinking direction for solving such problems, allowing the engineered bacteria to play the role of the "main character" with peace of mind, while leaving the tasks of colonization and protection to the "supporting characters".
During epileptic seizures, electroencephalogram (EEG) signals exhibit characteristics of spike waves, slow waves, and spike - slow complex waves. Clinicians typically rely on their extensive experience to make judgments, such as assessing the risk of epilepsy recurrence and evaluating the efficacy of antiepileptic drugs. Nevertheless, this process lacks a reliable, sensitive, and accurate machine - assisted detection approach. By extracting various features from the acquired EEG signals and performing analysis and calculations, it is feasible to assist clinicians in making more accurate and prompt diagnoses, thereby reducing the risk of misdiagnosis.
To provide such an auxiliary tool for the clinical diagnosis and treatment of epilepsy, we are dedicated to constructing an EEG - Based epilepsy disease detection and prediction model. Our objective is that for a detected and extracted EEG signal, once input into the model, the model can accurately yield a negative or positive judgment result for the input data.
We commence from electrophysiological signals, analyze the highly characteristic EEG signals during epileptic seizures, extract their temporal and frequency features, and utilize machine - learning methods for model construction. Using indices such as accuracy, precision, and sensitivity as evaluation criteria, we perform iterative cycles among different machine - learning models to seek the optimal solution of the model.
After being trained with machine - learning models such as Convolutional Neural Networks (CNN), Random Forest, and CNN - Long Short - Term Memory (CNN - LSTM), we have achieved good accuracy with an extremely limited amount of data. After 100 and 11,500 iterations, the accuracy of the model reached 80.88%, significantly enhancing its generalization performance. This optimized model serves as our final output.
Developing and training such a machine - learning model is of great significance for both practical applications and the update of modeling methodologies within the iGEM community. The achievement of high prediction accuracy with limited data is closely associated with model selection, optimization of training methods, and parameter adjustment.
In the future, we anticipate that more EEG signals that adhere to ethical and safety norms will be incorporated into the training, optimization, and iterative updates of this model. We also encourage more iGEMers to participate in our development efforts. Based on this model, they can conduct similar model development, thereby providing novel research perspectives for the field of neurological disease research.
The raw EEG signals extracted by the EEG equipment often contain a lot of noise, artifacts and other interfering information. To present more reliable and accurate EEG signals to the model, it is necessary to clean the original signals, such as removing artifacts and reducing noise. At the same time, visualizing this process and presenting the EEG readings over a period of time to the user is of practical significance, which helps to assess the risk of disease onset more purposefully.
Since the abnormal discharge of a single neuron is not sufficient to cause an epileptic seizure, the key lies in the synchronous activation of a large number of neurons, which is also the core mechanism of epilepsy. According to our research, in previous studies, most attention has been focused on the EEG analysis during the epileptic seizure process, while there has been little research on the abnormal discharge of a single neuron that is not sufficient to cause excessive synchronization of a large number of neurons. Therefore, we focus on this aspect and attempt to analyze the frequency of abnormal discharge behavior of a single neuron in the current stage, and then present it as a quantifiable risk index of disease onset. When the index exceeds a certain threshold, the user will be reminded to take medication in time. The ES: Detection software analyzes the data processed by EEG: QuickLab through model algorithms to realize our vision of an early warning system for epilepsy.
The design of this set of software is not only user-friendly, but we also adhere to the principle of open source sharing, which means that anyone can contribute and expand the functions of the software, enriching the ecosystem. Whether as a user or a developer, everyone can participate in the continuous update and iteration of this set of software. With the help of the GitLab platform, we can provide a brand-new EEG data processing software for the iGEM community and the world. In the future when more and more iGEMers participate in the development, we have reason to believe that such a set of software can be truly put into use, providing greater convenience for stakeholders.
The implementation of educational activities assumes a particularly significant role in the realm of synthetic biology. Science is inherently meant to be accessible to the public, comprehensible, and engaging, rather than being confined to a private domain, esoteric, and abstruse. Guided by this ideology, our educational initiatives have effectively forged a connection between the laboratory and diverse social groups. By bringing synthetic biology and iGEM to individuals of all age brackets, we have successfully facilitated knowledge dissemination and fostered an environment of exchange and sharing. During this process, our aspiration extends beyond merely imparting scientific knowledge verified within the laboratory setting. We aim to encourage a broader segment of society to actively shape, contribute to, and participate in synthetic biology research. This involves establishing a two-way dialogue with a wider audience, developing reusable educational tools, and promoting them comprehensively to expand the reach of education.
The optimization of educational activities has been a central focus of our endeavors. To achieve comprehensive educational coverage, we have introduced an innovative approach, conceptualizing and designing educational programs from three distinct dimensions: all-inclusive age demographics, diverse and innovative formats, and multi-tiered educational objectives. Based on this framework, we have developed an educational ecosystem cube. This serves as a valuable tool for evaluating and reflecting on each educational event, providing crucial guidance for subsequent initiatives.
When analyzed through the lens of all-inclusive age demographics, our educational activities span across various age groups, including children, middle school students, college students, the general public, and stakeholders. For children, our educational strategies emphasize the use of storytelling and interactive games. The content is carefully crafted to include life phenomena, scientific imagination within the context of synthetic biology, and fundamental biological concepts.
This integration of entertainment and educational content is designed to instill in young minds an appreciation for the wonders of science. For middle school students, the educational approach is more specialized. By integrating real-world case studies with hands-on experiments, we aim to convey the principles of synthetic biology and the significance of iGEM. Additionally, these programs serve as an introduction to scientific research and laboratory protocols, laying the foundation for future academic pursuits. In the case of college students, our activities place a greater emphasis on the social implications of synthetic biology. Topics such as disease awareness, emergency response skills, and social responsibility are incorporated to evoke a sense of civic duty and resonance among the students.
For the general public, we adopt a relatable and engaging approach. Leveraging short video platforms and live streaming services, we disseminate information on the practical applications of synthetic biology in daily life, health-related knowledge, behind-the-scenes insights into scientific research, and cultural connections. When engaging with stakeholders, we prioritize safety and support. Our activities are designed to promote positive interactions, reduce stigma, foster community support, and address health management concerns. These efforts not only facilitate a meaningful dialogue with stakeholders but also provide valuable feedback that guides the progression and refinement of our projects.
From the perspective of diverse and innovative formats, the utilization of live streaming, social media platforms, and interactive games has transcended traditional temporal and spatial limitations. These digital tools have established high-quality, accessible, and interactive educational channels, enabling the seamless dissemination of scientific concepts and values. Laboratory experiences, hands-on workshops, and cultural events provide a tangible and immersive learning environment. These activities not only reinforce theoretical knowledge but also infuse educational experiences with a sense of cultural and historical significance. The combination of traditional lectures with physical educational aids transforms the learning process from a one-way transmission of information to a dynamic two-way dialogue. This approach promotes an environment of equal participation and intellectual exchange.
In terms of multi-tiered educational objectives, our approach extends beyond the mere acquisition of knowledge and skills. We recognize the importance of cultivating emotional and attitudinal development as a fundamental aspect of education. By integrating synthetic biology, epilepsy research, and a variety of educational activities, we aim to inspire a deeper sense of empathy and social responsibility. This holistic approach fosters a learning environment where participants are encouraged to challenge existing biases and engage in meaningful discourse. As a result, our educational initiatives have facilitated a reciprocal exchange of ideas between our team and the audience, enriching both the learning experience and the development of our projects. The principle of "teaching benefits teachers as well as students," a timeless wisdom in Chinese educational philosophy, is vividly exemplified in our educational endeavors. The insights and feedback gleaned from these activities have not only enhanced the quality of our educational programs but also provided valuable impetus for the advancement of our research projects.
Reflecting on our educational achievements over the past year, we have significantly enriched the educational landscape within the iGEM community. Our innovative educational ecosystem cube offers a comprehensive framework for the design and evaluation of educational activities, encouraging a more diverse and inclusive approach to learning. Furthermore, the successful integration of games and cards into our educational curriculum represents a significant milestone. By combining entertainment and educational content, we have developed reusable educational tools that are both engaging and effective. We invite fellow iGEMers to join us in this noble endeavor of popularizing synthetic biology. By contributing diverse perspectives and innovative ideas, we can collectively sow the seeds of biological knowledge across the globe. With dedication and perseverance, we are confident that these seeds will germinate into a vibrant landscape of scientific understanding and innovation.
