Reviewing previous work in the field you choose to focus on is the first step in any project design. In this year's competition, we were able to verify two essential aspects of our project, namely, innovation and feasibility, from scientific literature research.

For one thing, we consider innovation as a project's ability to identify the gap present in a particular field and bring something new to make a difference. In our case, it helped justify our project in an age when new mechanisms, targets, drugs, and delivery systems were developed at an astonishing speed. Reviewing related literature on ASD treatment enabled us to focus on a more precise target, and put our efforts into developing a project for a specific condition. Besides, most of the literature we reviewed was recently published, which helped us keep abreast of the latest development in the therapeutic approaches to autism. It is also worth noting that we have attended several conferences on the cutting-edge studies of both the human mitochondria and ASD, thanks to the support from our PI, Professor Xingyin Liu. All of these made it possible for us to build a project that has underlying potential and is in line with the latest achievements in this field.

Feasibility, for the other, acts as an indicator of whether a project can be implemented in the real world. In order to better test the validity and feasibility of our project, we have looked at more than one hundred articles, many of which are from prestigious journals. We made sure that every decision we made, such as the choice of parts, chassis, and plasmids, has theoretical and experimental bases and has gone through thorough consideration.

You can learn about our research in terms of background and design in the Description and Design sections. Additionally, all of the references that we looked at are listed at the bottom of the related pages.

Wet lab work has been our major focus as we tried to demonstrate the feasibility of our design, allowing us to bridge the gap between theory and practice. We believe that getting down to real lab work and experiencing failures and success hands-on means a lot in the iteration of the project. In our 2022 project, we have carried out different types of experiments, including microbiological experiments, molecular biological experiments, as well as a series of other experiments, to verify the functions of our engineered bacteria. Our wet lab work started in July and lasted four months until October. Admittedly, we have encountered many difficulties as we went on with the experiments and the results we got were not exactly as expected. This doesn't mean, however, that we have been shaken out of the path even a little bit until the last minute. We have also organized weekly meetings where everyone shares their reflections on the experiment results. This allowed us to figure out the real problem and come up with a practical solution.

Our wet lab work for all the four systems in our design included at least one engineering design cycle. The good thing is that we have indeed got some exciting results and have validated our design little by little. We could feel a genuine sense of achievement when the bands that our plasmids ran on the gel turned out ideal and when a detectable shade of blue presented itself on the testing strip. Detailed protocols and experimental notes for each of our experiments are available on the wiki pages for Experiments. The results we got as well as our analysis, meanwhile, are recorded in the Results section. All of our failures and modifications are also documented on the Engineering page for future iGEM teams to refer to as they progress with their projects.

In terms of dry lab work, in this case, modeling, our efforts went from turning biological processes into mathematical languages to simulating the models on the computer. The nature of mathematical modeling enabled us to build specific models based on our needs and run the program as many times as we wanted, which helped us further prove our concept without using any reagents or laboratory equipment and provide a broader base for wet lab experiments.

Our dry lab work can be divided into two major aspects: structural analysis and functional analysis. We chose to carry out structural analyses because we believe that having an understanding of the structures of proteins, DNA, and other small molecules and learning how they interact with each other is important in synthetic biology. Specifically, we applied techniques such as homology simulation and analytical docking to visualize the molecules we were focusing on. In the meantime, we used ordinary differential equations (ODE), set parameters and variables, and simulated the functions of our gene circuits to explore the replication, transcription, and translation of genes. We were also able to get the inducer-target protein relationship with the data obtained from wet lab work.

You can learn about our models, their solutions, and related analyses in the Model section of our wiki.

Human practices should never be independent of the design, proof, and implementation of the project. We made sure that our human practices played a role at different stages of our project and gave us a better look at its influences on the world. As we organized our human practice activities, we sought inspiration and improved the project accordingly through INPUT activities such as interviews, questionnaires, and field trips. At the same time, we tried to spread the ideas, build connections, and reach a wider audience with the help of OUTPUT activities which include educational programs, lectures, and meetups.

Input

Understanding the experiences of stakeholders, experts, and family members of autistic people and getting their feedback about our project came first on our list. Together, we have organized five interviews at different stages of the project. In this process, we found support for the design, implementation, and overall goals of our project. In addition, in order to learn about the acceptance of synthetic biological products among our proposed end users, we designed a questionnaire, handed it out among parents of autistic children, and analyzed the results afterward. Three other questionnaires were also designed and analyzed to help us better target our educational activities. During the summer break, we also organized two field trips to the Children's Hospital of Nanjing Medical University and Star of the Sea, a rehabilitation center for children with ASD, which helped push our project forward.

Output

In this year's project, we also made an effort to educate and develop opportunities for individuals of all age groups and backgrounds in synthetic biology, ASD, basic skills in scientific research, and other subsets of biology. Our aim was to promote mutual learning experiences and encourage open dialogues among our audience, and all of us enjoyed sharing what we had known with people of different backgrounds. Besides, we valued collaboration and took the opportunity to participate in collaborations of various forms. We also hosted several activities so that other iGEM teams would hopefully benefit from them as participants.

Since the four systems in our project are all centered around mitochondrial activities and go from prevention to diagnosis and treatment, we have developed different implementation strategies for different systems. Specifically, for the lactate testing strip, we designed a piece of hardware. Regarding the two strains of engineered bacteria to treat mitochondrial dysfunction, meanwhile, we made a business plan to help promote their translated products.

Hardware

Hardware design helped us take our project from the laboratory to the real world. It should be noted that we decided to mainly design a piece of hardware for our lactate testing strip in System 4 and developed a lactate test kit since related regulations and laws remain to be further perfected in our country if we wanted to design a piece of drug-related hardware for the other three systems. Specialized knowledge of drug development will also be necessary, which was not easy for us to acquire overnight.

The lactate test kit is divided into three cells: the testing strip cell, the urine collection cell, and the safety cell. Besides, we designed a dropper to take in urine samples and, to safely store our chromogenic reagent, X-Gal, we developed a container as well. We tried to design the test kit to be good-looking, biologically safe, and lightproof. After building the initial model of the test kit, we 3D printed it for visualization. A user manual, or instruction manual, was drafted as well, presenting the principles of detection and the procedures for using the kit.

A detailed introduction of the hardware and its role in project implementation can be viewed on the Hardware page.Notice that different from our work in the implantation section, we went further to 3D printing and instruction-making at this stage.

Entrepreneurship

Countless ideas are generated daily, while only a tiny portion would develop into real products and enter the market. That means we will have to put in additional effort to implement our project in the real world. Commercialization, or in our case, translation, was the final step of the proof of our concept and was of great significance. Although the commercialization procedures may resemble that of other drugs since we focused on the therapeutic approaches, a considerable gap remains to be closed because we are engineering our probiotics.

Synthetic biology for therapeutic purposes is still viewed as a frontier in our country, with immature policies and ongoing discussions on related ethics and safety. Therefore, in terms of entrepreneurship, we first looked closely at existing laws and regulations and then carried out interviews with entrepreneurs at biotechnological companies to learn from their stories. With all that preparation, we were finally able to develop a business plan which included the design of our project, the analysis of the target market and our end users, as well as our evaluation plan. We understand that a qualified drug needs to go through a strict process, including animal experiments and clinical trials, before commercialization, and that cannot be accomplished within the season; however, we still hope that our efforts will support us as we keep exploring the application of synthetic biology in ASD treatment and offer more possibilities for autistic people and their families. You can learn more about our work in the Entrepreneurship section.

It should be noted that we have gone further to prove the concept based on our silver medal work of proposed implementation since we have contacted more entrepreneurs and experts and received credits regarding the business plans.