Before conducting laboratory operations, our team completed a biosafety ethics course and strictly adhered to the official biosafety guidelines provided by iGEM. Our experiments were conducted in a P1 level laboratory, and both the equipment and procedures complied with rigorous safety standards to ensure the safety of personnel and the environment. We selected safe strains of Escherichia coli, specifically DH5α, BL21, and Nissle 1917, and designed a bacterial suicide system (controlled by arabinose and temperature) to prevent gene leakage. We also prioritized privacy protection, ensuring informed consent from participants and data confidentiality. In summary, our experiments and data handling fully comply with biosafety, ethical, and legal requirements, ensuring the safety and compliance of the project.
Before conducting laboratory operations, our team undertook a biosafety ethics course. We strictly adhered to the official biosafety guidelines provided by iGEM, ensuring that we thoroughly considered the target user population, safety, potential for misuse, and the overall impact of our synthetic biology project. We understand that project design involves not only technical details but also social implications, laboratory safety, and ethical standards.
In terms of biosafety, we learned the importance of disease control, agricultural biosafety, and effective laboratory management. We ensured that potential risks to the environment and biodiversity were effectively controlled during our experiments. We strictly followed laboratory operating procedures, such as the correct use of alcohol lamps, clean benches, and autoclaves, to prevent occupational exposure risks.
Our team conducted rigorous safety assessments to ensure that the use of all engineered strains and related materials would not have a negative impact on the ecological environment or human health.
Our experimental work was conducted in a P1 laboratory equipped with standard laboratory devices and instruments, such as clean benches, refrigerators, oscillators, and centrifuges. The experiments were supervised by experienced instructors.
In our team’s laboratory work, we strictly adhered to comprehensive safety and hygiene management regulations. We ensured that all chemical reagents were returned to their proper places immediately after use, and tasting, drinking, or inhaling reagents was strictly prohibited. Any unusual behavior had to be reported immediately to laboratory management or the supervising instructor. After experiments, our team was responsible for cleaning up any waste and stains near the workstations, as well as performing a thorough cleanup, which included checking instruments, cleaning glassware, and returning chemicals to their proper locations.
If team members needed to leave the laboratory, prior consent from the team leader was required, and unauthorized departures were prohibited. During activities, members maintained a quiet and respectful demeanor, avoiding loud noises, running, or private entertainment activities. In the event of a violation, the safety officer had the authority to address the issue on-site. Students were not allowed to move between floors, take photos, or create noise, and access to the laboratory or other office areas outside of designated study times was prohibited. If we encountered any malfunctions or abnormal situations with the instruments, we promptly consulted experts or notified our instructors.
The laboratory was equipped with independent ventilation systems, and we were not permitted to open windows or adjust air conditioning without the scientist's permission. If environmental conditions caused discomfort, we were to report it and adjust according to instructions. Our experimental projects were all of high safety standards, and we adhered strictly to operating procedures to ensure safety.
Regarding the management of scientific instruments, we followed the principle of "do not touch unfamiliar instruments or press unknown buttons." In case of damage, we would be liable for compensation. Instruments used for timing, temperature measurement, pH measurement, and other purposes were not to be replaced arbitrarily, ensuring the accuracy and progress of our experiments.
For safety reasons, we selected the E. coli strains DH5α and BL21 for this project. Both DH5α and BL21 are commonly used laboratory strains of E. coli , widely applied in biological experiments due to their safety and specific genetic characteristics.
DH5α is a highly efficient competent cell often used for DNA cloning and transformation. Its features include antibiotic resistance and engineered mutations that enhance its stability and reliability in experiments. Importantly, DH5α does not carry any harmful viruses or pathogenic factors, making it relatively safe to handle under strict laboratory conditions.
BL21, on the other hand, is specifically designed for protein expression and is commonly used for recombinant protein production. The BL21 strain is genetically modified to lack endogenous proteases, allowing it to effectively express and accumulate foreign proteins without significant degradation by its own proteases. Similar to DH5α, BL21 does not harbor any pathogenic factors harmful to human health, making it a relatively safe experimental tool under standard laboratory operating conditions. These characteristics contribute to the widespread use of both strains in laboratories, ensuring the safety of personnel while complying with biosafety protocols.
Our ultimate goal is to develop a probiotic for weight loss, which is why we chose E. coli Nissle 1917 as our chassis microorganism. E. coli Nissle 1917 is an ideal strain for producing probiotics, primarily because it does not secrete intracellular toxins, reducing potential harm to the host while retaining the excellent characteristics of E. coli , such as rapid growth, ease of cultivation, and simple genetic manipulation. Therefore, Nissle 1917 is particularly well-suited for the development of edible engineered bacteria, enabling safe production of biomolecules and proteins.
In the field of synthetic biology, safety involves designing measures to prevent gene leakage.
The arabinose operon (ara operon) is a bacterial gene regulatory system that suppresses the transcription of related genes in the absence of L-arabinose. When L-arabinose is present in the environment, it relieves the repression on the pBAD promoter, activating the transcription of downstream genes. This mechanism allows bacteria to rapidly initiate their metabolic pathways when arabinose is available, effectively regulating their metabolic activities.
In our system, the introduction of the arabinose operon enables a safety mechanism for patients. If adverse effects occur or if the secretion of GLP-1 from the probiotic is not needed, patients can induce the bacteria to express lytic enzymes and initiate self-destruction by administering arabinose. This ensures that the probiotic remains under control and mitigates potential risks associated with its use.
Temperature Control System
Low-temperature inducible promoters are regulatory elements that activate gene expression under low-temperature conditions. These promoters are designed to be inactive at normal temperatures but significantly enhance their activity when the temperature drops. This mechanism allows for the transcription of target genes to be triggered in specific low-temperature environments.
In the event of strain leakage, the low-temperature inducible expression system will induce the expression of lytic enzymes when the temperature falls below 20°C, preventing contamination from the strain. This provides an additional safety measure to ensure that the engineered bacteria do not pose a risk to the environment or the host.
In order to ensure that the genes of the engineered bacteria would not be leaked, we linked arabinose operon and cryogenic inducible promoter with an "or" gate, which allowed the engineered bacteria to induce lysozyme expression under corresponding conditions to achieve the purpose of suicide (Fig 3A). First, we investigated the part library of iGEM, found arabinose operon pBAD(BBa_I13453) and low-temperature induced promoter pCspA(BBa_K4987003), and amplified the fragments (Fig 6B).
We measured the expression of red fluorescent protein induced by arabinose operon (Fig 4.A). With increasing arabinose concentration, the value of Flourescence/OD600 increases continuously, demonstrating that arabinose operon can induce the expression of corresponding protein through arabinose (Fig 7.B).
First, we verified the function of low temperature induced promoter. It was proved by experiments that the optimal growth temperature of E. coli DH5α was 37℃, and the growth of DH5α became slower as the temperature decreased (Fig. 5.B). We then introduced a low-temperature inducible promoter, pCspA, and attached a red fluorescent protein behind it to verify function (Fig. 5.A). The results show that the OD600 value of BL21 within 12h at low temperature is only 0.5, while the OD600 value of BL21 at 37℃ reaches 2.3. However, the Flourescence/OD600 value reaches 278 at low temperature, which is much higher than that of Flourescence/OD600 at 37℃ (Fig. 5.C), indicating that low-temperature induction of CspA can effectively induce the expression of red fluorescent protein.
We obtained two Lysozyme sequences of T4 Holin and T4 Lysozyme derived from T4 phage through the NCBI website and amplified them by PCR. We then genetically engineered arabinose operon pBAD and cryogenic inducer promoter pCspA to T4 Holin and T4 Lysozyme sequences. We separately verified the survival status of bacteria induced by two promoters. The results showed that in the presence of 0.5 mM/L arabinose, OD600 of the bacterial solution began to decrease significantly after 5 hours of culture, and reached 0 at the 20th hour (Fig. 6.A). However, at a low temperature of 16℃, engineered bacteria containing lysozyme sequences were always in a low density state, and OD600 was almost maintained at 0.3(Fig 6.B).
In summary, our team successfully constructed a suicide system with pBAD and pCspA as promoter, T4 Holin and T4 Lysozyme sequences as suicide genes, and successfully validated the function in E. coli DH5α species.
Privacy and safety are crucial as they protect personal information from unauthorized access and misuse, preventing identity theft and financial loss. Effective privacy protection not only safeguards individual rights but also enhances public trust in technologies and services. Therefore, we are responsible for taking proactive measures to protect personal privacy, including implementing stringent data protection policies and technical measures to ensure the security and confidentiality of information.
We have a responsibility to ensure that each participant provides clear and voluntary consent before participating in the research. This consent should include detailed explanations regarding the processing of personal information and privacy protection, ensuring that participants fully understand how their data will be used and handled. To achieve this, we need to communicate actively with participants, provide clear privacy protection information, and patiently address any questions or concerns they may have. Participants must be well-informed about how their personal information will be used and the measures taken to protect it. This transparency and communication not only help safeguard participants' privacy but also build the integrity and trust of the research.
To maximize participants' anonymity and the confidentiality of personal information, we should implement strict anonymization measures during data processing and sharing. For data involving personal privacy, such as names, addresses, or social security numbers, thorough anonymization is essential to ensure these data cannot be directly linked to individual identities. This means that when sharing data, all information that could reveal personal identity should be removed, and encryption or de-identification techniques should be employed to protect data confidentiality. Through these measures, we can not only protect participants' privacy but also maintain the security and integrity of the data.
The laboratory should establish a rigorous data management system to ensure that all data collection, storage, transmission, and processing comply with relevant laws and industry standards. The system should clearly define data access permissions, ensuring that only authorized personnel can access sensitive data. Furthermore, the laboratory should adopt advanced encryption technologies and physical security measures to safeguard data security. These measures not only prevent unauthorized access but also effectively mitigate the risks of data breaches or damage, ensuring the integrity and confidentiality of the data.
The research team and investigators have received appropriate training to understand how to handle and protect participants' personal information and privacy. All personnel should possess data security awareness, recognizing the importance of privacy protection, and consciously adhere to data management protocols in their daily work to avoid data breaches due to human error. This training and awareness development ensure that the team can effectively maintain participants' privacy, prevent potential security risks, and safeguard data security and confidentiality.
In conclusion, all experiments conducted by our team were carried out in a safe and supervised environment. Our data collection and investigations were completed in compliance with privacy regulations and Chinese laws. Overall, our team completed a biosafety ethics course prior to laboratory operations and strictly adhered to the official biosafety guidelines provided by iGEM. Experiments were conducted in a P1 laboratory that met safety standards, using E. coli strains DH5α, BL21, and Nissle 1917, and we designed a bacterial suicide system to prevent gene leakage. We placed a strong emphasis on privacy protection, ensuring informed consent and data confidentiality for participants. Our experimental and data processing practices strictly comply with biosafety, ethical, and legal requirements, ensuring the safety and compliance of the project.