1. Overview

JLU-NBBMS 2024 has designed a combination therapy that integrates bacteriotherapy of engineered bacteria delivering RNAi with nanoparticle-mediated chemotherapy. This strategy aims to reduce chemotherapeutic resistance in tumors and enhance the selectivity of chemotherapeutic drugs. We develop this strategy in order to enhance effective therapeutic responses and reduce adverse side effects induced by chemotherapy. Finally, it can lead to a significant improvement in treatment outcomes. While our research focuses on improving therapeutic outcomes, we also place a strong emphasis on biosafety and ethical considerations throughout the research process. The following is how we adhere to iGEM's safety rules and policies, as well as the measures we have taken to ensure the safety of our BIOTARGET project.

2. Design safety

2.1 Chassis safety

Molecular Biology Laboratory, College of Basic Medical Sciences, Jilin University possesses the delayed lysis Salmonella strain χ11802. To further reduce the virulence of the attenuated Salmonella and enhance the safety of the experiment, we knocked out the msbB gene.

To ensure the safety after our wet lab operations, we design the dry lab .Firstly, the msbB gene encodes an enzyme which is responsible for modifying the lipid A portion of bacterial lipopolysaccharides (LPS) through adding a lauroyl group to alter the structure of LPS. This modification reduces the bacteria's sensitivity to the host immune system therefore enhancing its survival. Deletion or mutation of the msbB gene increases the immunogenicity of LPS, making the bacteria more easily cleared by the host.Based on this, we can firstly assume that the endotoxin produced by the bacteria decreases. Additionally, we can assume that bacterial quantity is proportional to endotoxin levels. Referring to relevant literature and experimental data ,we can construct a mathematical model of the relationship between immune system changes and bacterial population dynamics.Through correlation analysis , we fit the trend of these changes and confirm the safety of Salmonella knocked out the msbB gene.

At the same time, we analyzed the function of delayed lysis strains and verified the safety of bacterial lysis .

Concurrently, the gene encoding T7 RNA polymerase has been inserted in situ to facilitate efficient expression of shRNA. The engineered Salmonella is capable of producing shRNA targeting chemoresistance genes via a T7 expression cassette, invading tumor cells. Besides, it releases the shRNA into the cytoplasm with the aid of listeriolysin O, a product encoded by the hlyA gene, ultimately activating the RNAi pathway to induce gene silencing. The hlyA is regulated by the anaerobically-induced promoter pnirB. The anaerobically induced gene expression and the tumor-targeting capability of Salmonella can restrict this process to tumor tissues, thereby enhancing the efficacy and safety of the treatment. When constructing these components, all DNA/RNA and plasmids utilized were carefully sourced through procurement, ensuring their safety and reliability.

2.2 Targeting module

We utilized the LPP OMPA bacterial surface display system to express the RGD peptide, which binds to integrins that are highly expressed in tumor tissues, thereby enhancing the specific targeting of attenuated Salmonella towards tumor tissues. Additionally, to enhance the therapeutic system's ability to local tumor and to adapt the system for various cancer types, the RGD peptide can be substituted with alternative tumor-homing peptides or antibody fragments. Furthermore, the environment of tumor tissues is more hypoxic compared to normal tissues. In this way it is possible to restrict hlyA expression to tumor tissues also to restrict tumor-specific trans-kingdom RNAi to tumor tissues, thus avoiding harm to normal tissues and ensuring safety.

2.3 Safety module

AThe strain we used is the delayed lysis Salmonella strain χ11802 that has undergone deletions of the asd and murA genes, rendering the mutant bacteria incapable of synthesizing a complete cell wall. Meanwhile, we have inserted the murA and asd genes into the companion expression plasmid, but the expression of these genes is under the control of the araCPBAD promoter. This means that the murA and asd genes can only be expressed in the presence of arabinose, thus ensuring the survival of the attenuated Salmonella. This bacterial system not only serves as a screening system for successful plasmid transformation but also ensures that when the arabinose concentration gradually decreases, the Salmonella gradually autolyzes after the Salmonella enters the body. In this way, it further guarantees the safety of the attenuated Salmonella.

3. Laboratory safety

3.1 Lab Safety Overview

When working on synthetic biology projects, we need to focus on safety issues at every step. Our team takes the safety of our members and the environment as a priority. Consequently, we are dedicated to taking all necessary precautions to avoid any personal injury or environmental harm.

Our team has refined laboratory safety guidelines, standardized experimental operations, and conducted systematic experimental operation training to jointly ensure experimental safety from various aspects.

3.2 Lab Safety

To prevent contamination, exposure, or accidental release during our experimental procedures, it is imperative that we ensure our laboratory facilities meet our requirements. You can find photographs of our working area below. Work was conducted across multiple spaces listed as follows.

Our laboratory is equipped with cylinder cabinets and gas cylinder storage cabinets, which are essential facilities for the safe storage of flammable, explosive, or toxic gas cylinders in the laboratory. Through the designs of fire prevention, explosion protection, robust structures, and ventilation systems, they can effectively reduce the risks of fire and explosion, facilitate management and reduce environmental pollution, thus ensuring the safety of laboratory personnel.

In the experiment, we handled biological materials with biosafety cabinets and chemical fume hoods.

We also possess advanced experimental equipment for precise experimental analysis and recording, which aids us in achieving optimal experimental results.

3.3 Training and Enforcement

In late March, all members of our team have received extensive safety and security training, covering key areas such as understanding laboratory access protocols.

We have systematically studied with instructors from College of Basic Medical Sciences, Jilin University, who possess extensive experience in laboratory risk management. We report any potential issues to our instructors initially, ensuring that our work is conducted appropriately. Additional containment measures are enforced as required to enhance safety. Moreover, we have developed specific policies for working alone and during off-hours, ensuring that a graduate student is always present or readily available. To further ensure safety, we have formulated communication plans and revised our experimental designs to incorporate biological containment measures.

Overall, we ensure that all work conducted is performed in a safe and controlled environment.

4. Safety in Integrated Human Practices

4.1 Personnel safety

Throughout Human Practices , we have consistently adhered to the principle of safety as the priority. At the beginning of the project, we visited relevant experts and companies to conduct a safety assessment of our project and signed safety agreements (see iHP). When entering biological laboratories, chemical laboratories, hospitals, production lines of enterprises, etc, we strictly adhered to relevant safety regulations, ensuring the safety of our team members while safeguarding the rights and interests of our supporters. Furthermore, we have compiled The Biosecurity and Bioethics Whitepaper that provides detailed explanations on various aspects of biosafety, serving as a reference for future igemers. We also hope that subsequent teams may continuously improve and expand this White Paper.

4.2 Personal privacy safety

We attach great importance to the personal privacy of stakeholders' during Human Practices. Prior to conducting a series of questionnaires and interviews, we have prepared detailed bilingual consent forms to ensure that the personal privacy of participantswon't be divulged. We pledge that all survey results and interview contents will be used only for purposes of statistics and research , and will not be disclosed to any third party or used for any commercial purposes. We have implemented a series of security measures to protect the personal information of participants, including encryption, access control, and secure storage methods, to prevent unauthorized data access, damage, or disclosure.

The content of the informed consent forms can be found in the attached PDF, which can serve as a reference for other igemers.