Our project makes several key contributions that can benefit future iGEM teams. We enhanced the kaempferol synthesis process using fusion enzyme technology by linking flavanone 3-hydroxylase (F3H) and flavonol synthase (FLS), improving the efficiency of the enzymatic reaction. We also contributed new data to part BBa_K3769002, demonstrating its ability to synthesize γ-Aminobutyric acid (GABA) from glutamate. In terms of biosafety, we implemented ultrasonic cracking to prevent gene leakage and selected Escherichia coliNissle 1917 as a safe chassis microorganism. Additionally, our human practices work provides recommendations for communicating with individuals with depression, offering valuable resources for teams working on mental health-related projects. These contributions provide practical guidance in enzyme design, biosafety, part improvement, and human practices for future iGEM teams.

Synthesis process of kaempferol: Naringin is catalyzed by flavanone 3-hydroxylase (F3H) to synthesize dihydrokaempferol, and then dihydrokaempferol is catalyzed by flavonol synthase (FLS) to form kaempferol. In this process, F3H and FLS are the key enzymes in kaempferol synthesis. We use fusion enzyme technology (adding linker in the middle of two enzymes to make the two enzymes close together to achieve the purpose of improving the efficiency of enzymatic reaction). We document in detail the design of the fusion enzyme, including the selection of linkers (e.g. linker length, type of linker) and how to verify the efficiency improvement of the fusion enzyme. By providing this innovative strategy, other teams can learn from our fusion enzyme design approach when designing multi-enzyme cascades.

We performed experiments on an existing GadB gene (BBa_K3769002developed by iGEM21_FZU-China). Our results demonstrate new applications for this component, as we have successfully verified that GadB can synthesize γ-Aminobutyric acid (GABA) using glutamate as a substrate. We have recorded the results of our experiments in the parts registry.

γ-Aminobutyric acid (GABA) is a key inhibitory neurotransmitter in the central nervous system, known for its water solubility, thermal stability, and safety as an ingredient in food and beverages. Due to its anti-anxiety and stress-relieving properties, GABA is widely used in the food and health supplement industries. Given the need for an edible, anti-stress, and anti-anxiety compound in our project, we initiated research into the production of GABA.

We first designed and constructed a GABA production strain. The GadB gene (BBa_K376900), encoding glutamate decarboxylase (GAD), was synthesized and codon-optimized for expression in E. coli. The gene was cloned into the pET23b plasmid using the EcoRI and XhoI restriction sites, generating the recombinant plasmid p23b-GadB. The construct was verified through sequencing, and the recombinant plasmid was extracted using a plasmid extraction kit. After verification, the plasmid was transformed into E. coli strains DH5α (for plasmid storage) and BL21 (for protein expression)(Figure 14).

Glutamate decarboxylase (GAD) uses pyridoxal phosphate (the active form of vitamin B6) as a cofactor to convert glutamate into GABA (Figure 15).

To catalyze GABA synthesis, we employed crude enzyme extracts from the engineered E. coli strains. The process involved the following steps: 1. Cell Harvesting and Enzyme Extraction: We collected 2 mL of bacterial culture, centrifuged it at 8000 rpm for 10 minutes to obtain the bacterial pellet. The pellet was resuspended in 2 mL of acetic acid-sodium acetate buffer (pH 4.6). This suspension was subjected to ultrasonic treatment (75 W, 1 second pulses with 3 second intervals, for a total of 20 minutes) in an ice bath to obtain the crude enzyme solution. 2. GABA Synthesis Reaction: In 1 mL of crude enzyme solution, 2% glutamic acid was added as the substrate, and the mixture was incubated at 37°C for 3 hours. 3. Quantification of GABA: After the reaction, GABA content was measured using a GABA test kit. The principle of the GABA test kit is that phenol and sodium hypochlorite react with GABA to produce a blue-green product, which has a maximum absorbance at 640 nm. Absorbance at 640 nm was recorded using a microplate reader, and a standard curve was generated to calculate the GABA concentration in the samples.

The linear regression equation is Y = 0.5130*X - 0.05257, with an R² value of 0.98. This indicates a strong linear relationship between GABA concentration and absorbance. Therefore, we conclude that GABA concentration can be reliably calculated based on absorbance.

BL21 and BL21/pET23b were used as control groups, while BL21/p23b-GadB served as the experimental group for the controlled experiments. By constructing the recombinant strain p23b-GadB, we successfully achieved GABA production, with the recombinant strain yielding 2.32 ± 0.21 g/L.

In our project, we prioritized safety by addressing gene leakage and the choice of chassis microorganism. These considerations are crucial for future iGEM teams working on projects related to food and beverage production.

Strain Inactivation with Ultrasonic Cracking: To prevent gene leakage, we plan to use ultrasonic cracking to inactivate our engineered bacteria. This method ensures that the bacteria are killed before they can be released, providing a non-chemical and efficient safety measure. Future iGEM teams can refer to this approach when seeking reliable ways to prevent gene leakage, especially in large-scale production processes.

Chassis Microorganism - E. coli Nissle 1917: We chose E. coli Nissle 1917 as our chassis microorganism due to its safety profile—specifically, its lack of endotoxin production. This strain has been widely used in probiotic applications, making it a safer option for projects involving human consumption. Our use of this strain offers a valuable reference for teams looking for a safe and reliable chassis organism in their projects.

These safety strategies, especially the use of ultrasonic cracking and a well-established chassis strain, provide practical guidance for future iGEM teams aiming to enhance biosafety in food-related projects.

Our project is aimed at people with depression and high stress. We summarized some precautions for iGEM team to communicate with depressed people in the future, as well as how to alleviate the anxiety of the interviewees. We hope our recommendations will help future teams who want to work on depression-related topics.

We have publicly shared all our project materials. In addition to these, after obtaining consent from the psychological counseling office and business teacher, we also uploaded their relevant resources. We hope that these materials will not only support future iGEM teams in the field of depression research but also provide valuable experience and reference for teams aiming to launch commercial projects.