Safety is one of the most important issues in research and experimentation. Especially for us, our program is designed for in vivo treatment in humans, and safety becomes even more important because it does potentially affect our health. Therefore, we actively take safety measures in laboratory safety and experimental safety to minimize possible hazards in our project. In addition, we also take into account the security of other people's information and protect other people's information from infringement.

Laboratory safety is very important to us. Before we enter the laboratory, we need to carry out laboratory safety skills training to ensure the safety of our experiments. In addition to this, we studied the experimental safety manual and the identification of possible hazards under the guidance of the lab safety faculty. These initiatives provide a solid foundation for our deep understanding of laboratory safety procedures and equip us with the knowledge and skills necessary to conduct safe experiments.

The materials we used in the experiment involve Escherichia coli Nissle 1917, DH10B, CT26, and MC38. All of them are included in the iGEM whitelist. We constructed plasmids and conducted bacterial experiments on BSL-1 benches, which are equipped with biosafety cabinets, eyewash stations, and ventilation systems to help us complete the experiments. For some cell experiments, we conducted them in BSL-1 laboratory, strictly abiding by the laboratory regulations, and all waste was disinfected.

Considering the potential cytotoxic effects of Tet v2.0, an inquiry was undertaken to ascertain its influence on bacterial survival by charting the growth trajectories of diverse bacterial species under a spectrum of induction regimes. The empirical evidence garnered from our study indicates that the cytotoxicity of Tet v2.0 is markedly less pronounced relative to arabinose and IPTG. The diminished growth observed in the F97 and S89 mutant strains is attributed primarily to the mutation-induced perturbations or the expression of a truncated form of CsgA. Consequently, our findings lead to the preliminary conclusion that Tet v2.0 exerts an inconsequential impact and exhibits minimal toxicity towards bacteria, thereby affirming its safety profile for application in experimental procedures.

Figure 1: Growth curve of bacteria and their comparison while expressing CsgA

Bacteria are cultured in 37°C within a microplate reader. After induction by Arabinose and IPTG, the population of S89TAG and F97TAG significantly decreased compared to other groups. Toxicity of Tet v2.0 was little.
WT: pBbB8k-CsgAWT

To further investigate the toxic effects of Tet v2.0 on animal cells, we conducted experiments to assess its toxicity towards the CT26 cell line. The experiment was designed with a control group (PBS only) and an experimental group (PBS supplemented with Tet v2.0). Cell growth was characterized by measuring the A450 absorbance, and the results indicated that the cell viability in the experimental group, with the addition of Tet v2.0, was significantly lower than that in the control group (normal cells). Thus, it can be concluded that Tet v2.0 exhibits considerable toxicity towards the cell line used in our experiments. This finding guides us to be cautious about the potential errors and impacts Tet v2.0 may cause in subsequent experiments and suggests the necessity to further engineer Tet v2.0 to reduce its cytotoxicity.

Figure 2: Toxicity of Tet v2.0 on CT26 Cells

Cells that had been cultured for a period were evenly divided into groups. The control group received an addition of PBS, while the experimental group was treated with PBS plus Tet v2.0, Using the CCK-8 reagent, cell viability was characterized by measuring the absorbance at a wavelength of 450 nm. The results demonstrated the toxic effects of Tet v2.0 on CT26 cells.

Considering the safety concerns associated with engineered bacteria and their secreted proteins, to minimize the potential for off-target expression of proteins in regions other than the intended lower rectal area, which could lead to unnecessary toxic effects, we capitalized on the hypoxic nature of the tumor microenvironment. By introducing a hypoxia-inducible promoter to control the localized expression of proteins, we aimed to enhance the safety profile of our engineered bacteria.

To explore the feasibility and expression efficiency of the hypoxia-inducible promoter, we utilized the promoter to drive the expression of GFP (Green Fluorescent Protein) for both qualitative and quantitative analysis. The results indicated that, in comparison to the wild-type control without the GFP gene (WT), the hypoxia-inducible promoter could indeed facilitate GFP expression under hypoxic conditions. However, there was a significant gap in expression efficiency when compared to the positive control group, which used an arabinose-inducible promoter, suggesting that the hypoxia-inducible promoter has relatively low expression efficiency.

This guides us to conclude that employing a new promoter can enhance the in vivo safety of engineered bacteria to some extent, but its low expression efficiency necessitates further design optimization for regulation.

Figure 3: Promoter Expression Efficiency Verification Experiment

Using the same culture and treatment methods, but employing different induction conditions: arabinose induction or hypoxic environment induction, and reflecting promoter efficiency by measuring the ratio of fluorescence value to OD600.

To prevent bacteria from leaking into the environment and causing harm to the environment, we designed a suicide switch. We selected the toxin protein MazF from the natural toxin-antitoxin system of Escherichia coli DH10B, a MazF protein sequence that is not present in the EcN genome. MazF protein can specifically recognize ACA sequences in single-stranded RNA. The most important cleavage site is ↓ACA, and sometimes it can become A↓CA due to the influence of upstream and downstream sequences.

We choose araBAD promoter induction MazF protein expression. This design helps us to improve our safety.

Figure 4: Suicide switch

We communicated with a number of professors and business executives. In order to ensure the security of information sources, we obtained the consent of information sources for every activity of human pracitce, so as to ensure that the rights and interests of others would not be infringed.

email from Professor Joshi

In order to comprehensively introduce and analyze the application of synthetic biology in the field of cancer treatment, We cooperate with JLU-NBBMS, XJTLU-Software, JLU-CP, SCUT-China-S, BNUZH-China, HKUST-GZ, OUC-Haide, SZU- China, BUCT-China, Guangxi U-China, SMU-China, UM-iGEM, SUSTech-Med, XJTLU-CHINA, NJU-China, SJTU-Software A total of 18 USTC-Software teams collaborated to write the bioethics white paper, which detailed the application prospects of synthetic biology in various track fields, ethical regulatory challenges, biosafety measures, project implementation details and case studies through multiple sections. The aim is to provide in-depth insights and references for researchers, policy makers, investors and the public in related fields.

First, through the track introduction plate, we clarified the key role of synthetic biology in cancer treatment, that is, by precisely targeting tumor cells, reducing damage to normal cells, thereby improving treatment effectiveness and reducing side effects. This section highlights the limitations of traditional therapeutic approaches and presents new ideas for using microbes as drug delivery vehicles, demonstrating the potential of synthetic biology to improve therapeutic accuracy.

Then, in the Bioethics/Bioregulation section, we discuss in depth the ethical principles and laws and regulations that must be followed in synthetic biology research and application. This includes key issues such as international and domestic guidelines, informed consent, privacy protection, and restrictions on data use. Through this section, we aim to ensure the legitimacy, ethics and public trust of research.

In the Biosafety section, we detail various biosafety measures and designs, such as self-limiting systems, environmentally sensitive switches, antibiotic resistance management systems, etc., which are designed to ensure the safety and controllability of experiments and prevent potential risks. Through these contents, we show the teams' awareness of the importance of biosafety, as well as their innovative thinking and technological accumulation in design.

The project introduction section focuses on the team's specific project, detailing the project's objectives, methodology, expected results, and how to evaluate the project against evaluation criteria. This section includes not only a detailed introduction to the team project, but also the team's general project and educational considerations, as well as the team's safety considerations and unique design features.

Finally, we demonstrate the practical application and potential impact of synthetic biology in the field of cancer treatment through case studies. By analyzing the "Gargantua" project developed by UPF CRG Barcelona 2018, we show how E. coli can be genetically engineered to absorb and degrade long chain fatty acids and reduce the metastasis potential of tumor cells. This case not only provides a novel treatment method, but also shows unique innovation and practicality in technology.

We hope to present the current status, challenges, innovative potential and future directions of synthetic biology in cancer treatment. By detailing the promise of synthetic biology in cancer treatment, ethical regulatory challenges, biosafety measures, project implementation details, and case studies, we aim to promote knowledge exchange, technological innovation, and policy development in the field of synthetic biology, while increasing public awareness and understanding of the field.

In summary, our various measures have ensured the safety of our experiments, with no incidents of danger or leakage occurring. We are firmly convinced that our approaches have successfully safeguarded us against any potential hazards.