Part 1: Public

After conducting background research, we designed a relevant questionnaire to determine the public's awareness of synthetic biology and gut-colonizing engineered microorganisms. This questionnaire was given to people from various backgrounds and age groups. Besides studying the public's understanding of synthetic biology and gut-colonizing artificial bacteria, it also included questions about gut health knowledge and the impact of lifestyle behaviors on gut health.
We subsequently received more than 200 responses. According to the data analysis shown in the figure below, the public's understanding of synthetic biology is still superficial, their awareness and acceptance of gut-colonizing engineered bacteria are quite low, and their knowledge of the impact of lifestyle behaviors on gut health is even lower.
In response to this circumstance, we designed a variety of educational events aimed toward various age groups to broaden public awareness in these areas.

Part 2:Academic

Cleavage Efficiency

This year, we designed a delivery system for our project Intestide that allows bacteria to display small peptides on their surface. For the short peptide to enter the gut and start working after enterokinase cleaves it, we included an enterokinase cleavage site before it. However, due to the complicated environment on the bacterial cell membrane and the short length of the peptide chain we wanted to express, the cleavage site may not interact well with the enterokinase, making high-efficiency cleavage difficult. The WHU-iGEM team chose to meet with Professor Yingliang Wu, a specialist in proteins, to improve our experimental design even further.
First, we questioned about the cleavage efficiency of our current design. In response, Professor Wu stated that we need to first identify enterokinase's active location. The cleavage efficiency will be higher if the enzyme's active site is visible on the surface. On the other hand, the cleavage efficiency will suffer if the active site is internal. He suggested placing a flexible linker between the enterokinase site and the transporter protein to increase the possibility of interaction with the active site to solve this problem. To enhance our experimental results further, we could investigate the connection between linker length and cleavage efficiency.
We then inquired as to how we could verify the cleavage efficiency. Professor Wu suggested that to determine the outcome of the cleavage, we might consider separating the bacterial cells following cleavage, purifying the cleavage products from the supernatant, and using techniques like mass spectrometry and HPLC.
Lastly, Professor Wu offered an invitation to his PhD candidates to take on particular experimental queries about chassis choice and induction settings. We discovered that BL21 Rosetta has a greater number of uncommon codons and is hence more suitable for expression vectors than the widely used expression vector E. coli BL21. In terms of induction conditions, we discovered that 25°C for 12-16 hours and 37°C for 6-8 hours are both acceptable.

Experiment Design and Plasmid Contribution

For experimental design, instrumentation, and plasmid construction in microbial synthetic biology, the WHU-iGEM team still had many questions. Therefore, we visited Professor Fei Gan from the Department of Microbiology at Wuhan University to seek her guidance.
Professor Gan points out that our engineered bacteria may not be able to survive in the complicated gut environment, and that the dynamic changes in the gut may make it more difficult for our complicated system to function. Using strong promoters (like T7) could cause the engineered bacteria to give all of their energy to protein expression, which would slow growth, and this is another thing that should be taken into consideration as well. She suggested that we could design our engineered bacteria to gain a competitive advantage, helping them to survive and colonize the gut.
About plasmid building, she suggested that we compare our constructions to commercial vector sequences and answered some of our inquiries concerning protein tag selection and amplification strains. She gave us a brief description of the equipment and associated viability of the experiments at the School of Life Sciences, including their capacity for fluorescence analysis and cell sorting.
Professor Gan provided several ideas for experimental design, including using Western Blot or chromatography for quantitative protein analysis and pure GST as an internal reference. We could perform cell membrane extraction if we were experimenting with membrane proteins. She also stated that the best period to induce protein expression is usually during the logarithmic growth phase and suggested conducting time gradient tests while keeping protein leakage in mind.

Micromolecules Function

To investigate the function of micromolecules in the gut, we visited the expert in this field, Professor Chang Jiang Dong who is from the School of Pharmacy at Wuhan University.

Professor Dong recommended that we calculate the number of transmembrane domains in the protein to ensure optimal transmembrane placement. To accomplish optimal expression and presentation, we need also to evaluate the properties of Gram-negative bacteria's inner and outer membranes, as well as review relevant research publications.
Furthermore, since the experiment could be used in the gut in the future, Professor Dong recommended we need carefully select the E. coli strain to ensure its survival in the intestinal environment. He also suggested identifying the polypeptides to be used in the project and increasing detection effectiveness through methods such as immunofluorescence and enzyme digestion.
Lastly, Professor Dong encouraged us to investigate further bacterial display systems while considering short peptide absorption. He suggested integrating antimicrobial peptides, which can prevent a variety of diseases and have promising application potential.

Feasibility of Project

To ensure the feasibility of our project, we visited Mr. He Xing Xing the who from Wuhan University School of Medicine. Professor He Xingxing stated after looking over the project's general design that although the project's concept is a possibly viable approach for the future, there is still a long way to go before it can be completed. He offered the following helpful recommendations:

1. The Limitations of Short Peptides
Professor He pointed out that since short peptides are small molecules, they can be difficult to absorb in the complicated environment of the gut. Furthermore, studies on short peptides are still in their early stages, and there is little proof of their therapeutic or other benefits. He emphasized that before moving the initiative further, more research on short peptides must be done.

2. The Limitations of Engineered Bacteria
Professor He pointed out that the delivery of drugs through engineered bacteria is not yet a reliable technology. The dosage of medications must be tightly controlled, yet the bacterial population in the gut can fluctuate due to the complicated environment, making drug release unpredictable.
Furthermore, the gut ecology differs from person to person, therefore modified bacteria may not be suitable for all persons. The same strain may not work for everyone.
He also cautioned that synthetic bacteria could disturb the gut's natural microbiota, perhaps leading to digestive or associated illnesses. Even though our design includes a module to manage bacterial density, we still face considerable problems. Questions such as how to accurately manage bacterial population and peptide release concentration, how to modify bacteria for various persons, and how to ensure safety are crucial areas for future research.

3. The importance of providing short peptides as supplements
We suggested that short peptides might be used not only as supplemental medicinal agents but also as dietary supplements to provide critical amino acids for growth and disease prevention. However, Professor He emphasized that, with the advancement of synthetic industries, the creation of numerous supplements is now extremely advanced and economical. Engineered bacteria, as genetically engineered organisms, are likely to cost more than existing products. Furthermore, the general public is wary of consuming genetically modified food, and most people are unlikely to pick altered bacteria as a nutritional source. He proposed that we focus our efforts on lowering costs, increasing product accessibility, and developing strong reasons to replace current supplements.

Part 3: Enterprise

Application

Dr.Mei Ye
We must have in-depth conversations with stakeholders to fully understand how our system is implemented in practice. To further investigate the project's potential applications in medical fields, the WHU-iGEM team have a speak with Dr.Mei Ye, a specialist in gastrointestinal disorders at Wuhan University Zhongnan Hospital.
The following recommendations for our project were given by Dr. Ye:
1. Design of the System:
Drugs for the intestines can function in two ways: either by functioning within the intestinal lumen or by entering the bloodstream. It is important to clarify the exact way of action of our short peptides, which mainly function via circulation.
2. Prospects for Application:
Adherence to Medication: Individuals might forget to take their prescribed medication, and the irregular amount may hurt their quality of life. It makes sense to take medications less frequently. Pharmaceutical attributes: A good medication should be inexpensive, simple to take, safe, fast-acting, and effective. We must take into consideration the drug's safety and possible negative effects, as well as its ability to be released, take effect, and sustain its efficacy if we desire to convert our research into useful outcomes.

Dr. Wang Li Ting
To find out how to implement our project in the field of child treatment, we visited with Dr. Wang Li Ting, the director of the pediatric rehabilitation department.

Dr. Wang provided detailed answers to our questions and offered valuable suggestions for our project:
●Does expressing AQ in our bodies help or support the treatment of illnesses like dwarfism?
○AQ is rarely used in treatments. Currently, patients with short stature must inject medication daily or monthly, and the type of medication used varies. There is no single, effective treatment available, for example: some medications stimulate height growth by increasing sleep, but their efficacy is unclear and lacks large-scale evidence. One modern injectable drug is growth hormone, a protein. If an oral drug could be created, it would be a godsend for children of short height who require long-term injections, as they are frequently terrified of and resistant to them.
●Is long-term medication and nutrient supplementation (especially for the elderly and children) a complicated or unresolved issue in clinical practice?
○If the short peptides for treating diabetes and hypertension are found to be safe and effective, this initiative will be extremely beneficial to senior individuals. Childhood diabetes is a relatively infrequent condition. Capsules are simple for adults to take, but for children converting them into a powder or dispersible pill may be more appropriate and easier for them to take.
●What modifications or targeted research are required to transform our idea into an achievable outcome?
○My recommendation is not to add new functions to the system, but to broaden the product offering. Creating a variety of modified microbes targeted to specific illnesses would make promotion easier and reduce product costs. Furthermore, this strategy would reduce the use of other treatments, making the products more attractive.
●What are your thoughts on the feasibility of our project?
○I believe the overall premises of your project are sound. Probiotics research is currently popular, and in the future, many diseases such as diabetes and hypertension may be cured with probiotics. There's no need to be too concerned about gut microbiota difficulties. Most people would choose native strains first, then cultivated non-native strains (which may be pathogenic). Probiotics are currently in widespread use in clinical practice, specific probiotics are used in children to treat autism, depression, sleep difficulties, and cognitive impairment. Bifidobacterium infantis, a popular pediatric probiotic, is combined with three other probiotic strains and supplied in a milk powder basis that children readily absorb.

Dr. Wendy Tan
On the other side, we also interviewed Dr. Wendy Tan who joined as clinic director at Vanfo Hino Clinic.On the other side, we also interviewed Dr. Wendy Tan who joined as clinic director at Vanfo Hino Clinic.

●Following our explanation of the project, Dr. Wendy praised those therapy applications that are sustainable.
●She advised us to keep our sights on the intestinal short peptide's slow release, she stated that in therapy, peptide release needs to be as gradual and controlled as possible.
●She also indicated that vaccines might be a new application of our project. In some new types of vaccines, peptide molecules could be utilized as carriers to transport viral proteins into the body, displaying the viral proteins as antigens and stimulating the immune system to respond.
●Dr. Wendy approves the application of the project's modified E. coli bacteria in the gut while E. coli bacteria originated in the intestine microorganism's environment and return to the intestine microorganism population without causing a rejection reaction.

Hunan Bizu Biotech Company
Hunan Bizu is a China biotech company that provides stem cell and immune cell storage, and also biotransformation medical technology services.

Here are the advices given by Mr. Jiang:

●I think there's a good chance this project will work if you go for the food-grade market, more especially the dual-purpose health supplement market (dietary and medicinal). As far as I know, most teams are working on polypeptide release, such as our company, which creates polypeptides to treat Parkinson's illness.
●You should concentrate on determining if the short peptides are produced continuously and how this release impacts the gut microbiome. If the project is carried out, it will have a major impact on the future application of the product.
●The human central nervous system is greatly influenced by the gut microbiota, even though your research may not be directly tied to the "gut-brain axis" (microbiome-gut-brain-neural system). Many medications for mental illnesses are designed to impact the gut microbiome. Understanding this could be crucial for your project.

CytoHealth
CytoHealth is Malaysia's pioneer Research and Development Bioprocessing company in the field of Regenerative Medicine and Immunotherapy.

We had an online conversation with Ms. Pei Jun Chai, the leader of one of the research teams, and she provided us with many pieces of advice:
●Identify a particular medical need or application for the product.
●Market Research: Identify underserved medical needs or possibilities where peptide-based treatments can help. This could include conditions like inflammatory bowel disease (IBD), gastrointestinal(GI) infections, metabolic problems, or gut health.
●Target disease or condition: Choose a disease or condition that would benefit from peptide therapy. For example:
○Peptides for treating IBD, such as anti-inflammatory peptides.
○Antimicrobial peptides (AMPs) for gut infections.
○Peptide-based enzyme treatment for celiac disease or lactose intolerance.
○Peptide vaccines or supplements for GI health.
●For in vitro or in vivo studies to evaluate the peptide's bioactivity, stability, and absorption, the peptide must be more accurate in hitting the target.
●List the advantages of short peptides in comparison to nanoparticles.
●Differences in efficiency might be related to the product's form (powder or capsule).