1. 1.We are not using any organisms from Risk Group 3 or 4.
2. 2.We are not releasing or deploying a genetically modified organism outside the lab.
3. 3.We are not testing our product on humans.
4. 4.We are not working with animals or samples from animals.
5. 5.We are not bringing a product of a genetically modified organism outside the lab.
6. 6.We are not conducting laboratory experiments using human samples, such as blood, DNA, other bodily specimens, and health or psychological outcomes.
7. 7.We are not using parts or organisms obtained from anywhere other than a trusted commercial or institutional supplier.
8. 8.We are not biasing the inheritance frequency of a genetic marker in an organism's progeny, i.e. creating a gene drive.
9. 9.We are not Increasing risks from antimicrobial resistance, such as by using novel resistance factors.
10. 10.We are not doing surveys, interviews, or other human subjects research.

Saccharomyces Cerevisiae is in Risk Group 1 but not on the White List. We realized that there was a problem with the Preliminary Project Safety Form and that we needed to fill out a check-in form to be excluded from the White List, so we filled out the check-in form to apply for permission to use and to learn about the potential dangers of Saccharomyces Cerevisiae. We have standardized our experimental procedures and operations to minimize the risk of Saccharomyces Cerevisiae as much as possible in our experiments. Our check-in form for the Saccharomyces Cerevisiae application has now been approved.

We conducted all the experiments in the 3M Lab of Nanjing University and the Institute of Functional Biomolecules of Nanjing University. Both labs had several master's and doctoral students to supervise our experiments, as well as several supervising professors to supervise and guide our experimental design. Both laboratories are fully equipped with instruments, safety management measures, and clean and safe experimental environments. We performed all microbiological operations in the aseptic bench. The two laboratories are supervised by Chenyu Zhang, Dean of the School of Life Sciences at Nanjing University, and Huiming Ge, Associate Dean of the School of Life Sciences at Nanjing University, and have high laboratory safety specifications.

Lab

Lab

Ultra-clean bench

1. 1.In terms of human health or safety hazards, prolonged exposure to high concentrations of yeast spores or cellular residues during yeast transgenesis and culturing may lead to allergic reactions to yeast in laboratory personnel, especially those sensitive to yeast cell wall components. During electrophoresis, the experimenter may come into direct contact with toxic electrophoresis dyes, so we will wear nitrile gloves. When conducting larger-scale yeast cultures, yeast spores may be inhaled by the experimenter, causing allergic reactions.
2. 2.In terms of environmental hazards, S. cerevisiae or its spores can be transmitted through the air, equipment, or laboratory personnel, resulting in cross-contamination of other experimental materials or cultures in the laboratory. The use of genetically modified S. cerevisiae may lead to problems of gene leakage in the external environment, affecting the ecological balance or causing undesirable consequences. In large-scale yeast culture, yeast spores may spread with the air and enter the environment outside the laboratory, causing environmental pollution.
3. 3.In terms of other hazards, when picking out colony monoclones or inoculating yeast on culture media, the inoculation needle may accidentally cut into the skin of the experimenter and cause injury. Alcohol lamps are used to sterilize bacteria in ultra-clean benches, which may cause burns.

Our project addresses risks related to yeast leakage, chemical reagent safety, and injuries from sharp objects in the laboratory. Our team has completed cell biology and molecular biology courses, mastering standardized experimental techniques such as plasmid construction, transformation, and bacterial strain cultivation, as well as using essential instruments like centrifuges, oscillators, PCR machines, and ultra-clean workbenches.

Prior to laboratory work, our supervisor and PhD students provided training on instrument operation and safety protocols, including bacterial strain disinfection. We adhere to strict safety measures: wearing nitrile gloves when handling chemicals, masks and gloves during Saccharomyces cerevisiae experiments, and conducting work beside alcohol lamps on ultra-clean benches. All equipment in contact with yeast is treated to minimize leakage risks.

We also follow lab safety regulations diligently. Most yeast experiments are performed in sterile laminar flow hoods, ensuring proper ventilation. Post-experiment, all lab personnel and materials undergo thorough sterilization to prevent yeast escape from the lab. Additionally, lab wastes and disposable items that come into contact with yeast are disposed of in bio-waste sorting bins, further mitigating potential risks.

In our scenario, yeast with high resistance to temperature, oxidative stress, high/low osmotic pressure, etc. can be better adapted to unfavorable environments in production environments where yeast is used for industrial production, production of food, and production of everyday products such as medicines, biofuels, bread, beer, etc. Increasing the productivity of yeast and potentially having the ability to bioproduce in situations where bioproduction would not otherwise be appropriate, replacing chemical production, reducing the use of chemical additives and chemicals that are detrimental to the environment or health.

Our highly resistant yeast has the potential to enhance productivity and reduce chemical use in food and industrial production. However, in poorly sealed environments, there is a risk of yeast coming into contact with personnel or producing airborne spores that can escape into the natural environment. The yeast's superior resistance may give it an advantage in the field, potentially disrupting local microbial balances. Additionally, there is a risk of gene migration, where the TPS1 gene could transfer to other bacteria, increasing their resistance and potentially harming local flora and fauna.

But this is only our preconceived idea, our actual goal is to help us realize our preconceived idea through our own constructed AI model, and to successfully prove through experiments that the AI model is practical and has the ability to help people realize the idea of synthetic biology, without the idea of actually putting it into production. Thus corresponding to the next question in the safety form, after the competition is over, and in our future plans, we do not intend to further develop our highly resistant yeast, even though we envisage a scenario where the yeast would need to be released outside of the lab environment, and the highly resistant yeast that we construct in the lab would be limited to simple authentication of our AI model only during the course of the competition, and after the competition.

Future risks primarily involve environmental contamination and ecological crises stemming from yeast leakage during production. We consulted with staff from Loxi (Shanghai) Medical Technology Co., Ltd. and Mr. Shi Junfeng, Project Director at the Eye, Ear, Nose, and Throat Hospital of Fudan University, regarding the ecological implications of such leakage. They advised that we should overexpress only a few key genes, specifically TPS1, to minimize the impact of gene migration and drift, as alginate synthesis pathways do not exist in other strains. This approach reduces the risk of unintended expression.

Despite the low risk associated with Saccharomyces cerevisiae, which is our source organism, it is essential to implement robust sealing measures for any future bioproduction. We plan to carefully draft usage instructions, emphasizing sterilization and disinfection to prevent leakage of the engineered yeast. Additionally, we will inform potential future users of our yeast about these safety measures.

Safety meeting