Our research is intimately connected with the human body, making biosafety a critical concern. Biosafety risk encompasses the potential threats that organisms or biological substances may pose. Consequently, it is imperative to implement preventive measures against these risks.

In addition to managing toxic reagents and laboratory activities, particular care is taken when handling bacteria that present a significant hazard. Therefore, we implemented rigorous experimental design and comprehensive training to ensure safety.

We used the specific attenuated strain VNP20009 due to it being highly attenuated. VNP20009 is unable to synthesize purines requiring a purine rich environment to survive and replicate, limiting its capacity for pathogenicity or viability outside very specific lab conditions and in vivo conditions like the tumor microenvironment. This limits many risks typically associated with working with bacteria.(Clairmont et al., 2000)

Our plan is to deliver Salmonella into tumor cells. This involves introducing Salmonella into the human body, necessitating strict control over its population to ensure the risk to the human body remains within a manageable range.

Inspired by M. Omar Din's literature Synchronized cycles of bacterial lysis for in vivo Delivery, we designed a synchronized lysis circuit (SLC) to control Salmonella's population level.

φX174E, also known as lyseE, is a gene that codes for the E protein of the Phix174 microvirus (Sinsheimervirus), which induces host cell lysis. The circuit includes a common promoter, pLuxI, which drives the expression of downstream genes. AHL, an autoinducer produced by pLuxI, binds to LuxR and transcriptionally activates the promoter, thereby inducing the expression of target genes.

AHL diffuses to neighboring cells, providing a mechanism for intercellular synchronization. Consequently, the concentration of AHL can reflect the size of the bacterial population.

The bacterial population dynamics regulated by this system can be conceptualized as follows: when AHL proteins accumulate to a specific threshold, bacteria initiate large-scale lysis, leading to a rapid decrease in population density. Following lysis, a small number of surviving bacteria resume AHL production. (Din et al., 2016)

To minimize the risk of potential cytokine storms and inflammation, we selected the BAX gene, a pro-apoptotic member of the Bcl-2 gene family, as our killer gene. The BAX protein is reported to increase the opening of the mitochondrial voltage-dependent anion channel (VDAC), leading to a loss of membrane potential and the release of cytochrome c. When cytochrome c reaches cytotoxic levels in the cytosol, it activates cysteine proteases, ultimately inducing programmed cell death. In this process, the fragmented cell components are encapsulated by apoptosis bodies, preventing damage to neighboring cells. Additionally, in normal cells, the BAX protein primarily exists as an inactive monomer in the cytosol or occasionally at the mitochondrial membranes. This means, the expressed protein does not typically damage the adjacent cells normally.(Zhang et al., 2017)

Since activated Bax protein may have risks in inducing apoptosis in normal cells as well, we utilized STIFs to manipulate the mRNA circularization process to control its expression.(Shao et al., 2024)

An expression vector was designed to produce a synthetic mRNA transcript containing a control region with RBP-specific aptamers in the 3'-UTR, specifically MS2-box motifs. To minimize BAX mRNA translation independent of STIFs, trans-acting shRNA-binding sites were replaced with cis-acting hammerhead ribozyme (HHR) motifs, which induce spontaneous self-excision of the natural poly(A) signal. An antibody linked to rotaviral non-structural protein 3 (NSP3), which is an eIF4F-binding protein (eIFBP), recognizes the intracellular signal, while another antibody recognizing the same signal is linked to bacteriophage-derived MS2 coat protein (MCP), which binds to tandem repeats of cognate MS2-box motifs. These plasmids form a killing system activated by STIF. Only when the two antibodies recognize the specific signal, forming a loop with BAX mRNA connected to NSP3-antibody-signal-antibody-MCP, translation of BAX protein will occur to induce cell apoptosis of cancer cells.

To strengthen the safety of university laboratories, ensure the personal safety of teachers and students, and maintain campus stability, the Ministry of Education of the People's Republic of China has issued the Safety Standards for Higher Education Laboratories and distributed them to all schools.

Our school also places significant emphasis on experimental safety and has established numerous regulations for experimental operations and instrument management.

To qualify for entry into the lab, each member of the wet lab team must watch educational safety videos and pass the laboratory safety examination. The exam covers electrical safety, hazardous chemicals, biosafety, the use of special equipment, fire protection, and first aid. Only individuals who have obtained a certificate of passing the laboratory safety examination are permitted to enter the lab.

Dr. Yanbo MAO serves as the guiding teacher for our project, and along with Dr. Jia YOU, they also teach our experimental class. Both are well-versed in molecular and cellular experiments and can offer guidance at any time. During our laboratory sessions, the two teachers introduced and emphasized the importance of laboratory safety protocols, ensuring our experiments are conducted under their supervision.

According to the safety guidelines issued by the World Health Organization, Salmonella requires a safety level 2 designation, necessitating that experiments be performed in a P2 laboratory. Four members of our wet lab team have obtained the P2 laboratory admission certificate: Chenlu Xue, Dingqi Gu, Weiyan Cheng, and Yishan Zhou. Only members who have obtained the certificates are allowed to enter P2 laboratory.

In 2019, the Center for Excellence and Innovation in Brain Science and Intelligent Technology of the Chinese Academy of Sciences released P2 laboratory safety specifications. When conducting experiments involving Salmonella, our researchers strictly adhered to these requirements, despite VNP20009 being an attenuated Salmonella strain with low pathogenicity. For instance, experimenters in the P2 laboratory wear two layers of gloves during operations, don disposable surgical gowns, and sort and dispose of various waste materials accordingly.

To ensure the safety of teachers and classmates, numerous safety facilities are installed in the laboratory, including eye wash stations, fire extinguishers, first aid kits, fume hoods, and sterilization equipment. Additionally, the laboratory is well-stocked with lab coats and gloves. These precautions are essential to mitigate the risks associated with exposure to harmful substances during experiments.

Before bacteria are officially utilized, our team will conduct rigorous safety testing in accordance with the requirements of the national government and relevant departments. We will also optimize the process under professional guidance to ensure that the bacteria and related biological components pose no harm to humans or the environment.