Safety Work
In order to ensure the safety of experimenters, the environment, and future users of this project's products, safety has always been one of the most important criteria for evaluation in this project. Therefore, we have adopted extremely high safety standards throughout the project design and experimental operations. The safety of this project is mainly reflected in four aspects: product design, experimental process, environmental impact assessment, and user safety. To ensure the safety of the above aspects, we conduct comprehensive risk assessments before each link, establish effective management measures, and formulate detailed emergency response plans. We also ensure that each participant must master the relevant safety regulations and pass the safety assessment before performing related operations. Our safety standards are formulated based on the iGEM safety guidelines, national laws and regulations, and other professional regulations. We also have consulted professionals and experts in the field of biosafety. The above measures guarantee that our project is in a safe and controllable state all the time. In this section, we will elaborate on our efforts to ensure safety.
I.Compliance with iGEM Rules and Policies
This project strictly complies with the rules and policies of iGEM and refrains from any activities prohibited by iGEM. We are clear about the iGEM whitelist, and all organisms, parts, and related activities involved in the experiment are included in the whitelist. We only use mice as experimental animals and have obtained permission from iGEM. When conducting human subject research, we have confirmed that the information collected does not require us to obtain approval from relevant laws, regulations, and institutional rules. We ensure that the collected information is not used for purposes other than research and and remains confidential, with no disclosure to the public..
II.Safety of Product Design
Since the goal of this project is to develop a safe engineered bacterium for the treatment of IBD, we must consider the risks that the engineered bacterium may pose to the interaction with the body of IBD patients during the product design phase and design corresponding control methods.
Risk Identification:
1.Infection risk: The engineered bacterium itself may pose an infection risk.
2.Drug toxicity: The drug molecules carried by the engineered bacterium may be toxic.
3.Incorrect localization: The engineered bacterium may express adhesion proteins and colonize in unexpected locations (such as the mouth), causing the drug to be ineffectively released.
Risk Management:
1.Use of safe biological tools: We choose Escherichia coli Nissle 1917 (referred to as EcN) as the engineered bacterium because it is the only non-pathogenic E. coli. EcN also has a protective effect on the mucosa and is commonly used to treat IBD.
2.Control of drug toxicity: The drug molecules we use are peptides, which can be broken down by the human body. Although both drug molecules have certain cytotoxicity, our designed regulatory elements can control the expression level of the engineered bacteria. We have also determined the appropriate dosage through experiments to control the colonization of the engineered bacteria in the intestines, which allows us to maintain the drug concentration within a safe range to avoid adverse effects on the human body.
3.Design of regulatory elements: We have designed an oxidative stress promoter, a regulatory element that can regulate the expression of drug molecules according to the level of reactive oxygen species in the intestine. The level of reactive oxygen species is positively correlated with the severity of IBD. Under the control of this regulatory element, the higher the level of reactive oxygen species, the higher the expression of drugs. In the low reactive oxygen species conditions of a healthy intestine, the response level of the promoter is not high, and the expression of drug concentration is significantly reduced.
III.Safety of Experimental Process
A safe experimental process protects the personal safety of every project participant and is also an important guarantee for the rigor of the experiment. To ensure the safety of the experimental process, we mainly consider the following aspects:
1.Professional consultation: Before the start of the project, we consulted professors with rich experience in biosafety and related fields. All operations in this experiment have been approved by the relevant professional heads.
2.Laws and regulations: All our experimental activities comply with relevant laws and regulations such as the "Biosecurity Law of the People's Republic of China" (2020), the "Regulations on the Management of Laboratory Biosecurity of Pathogenic Microorganisms" (2018), etc. In addition, we also comply with the "Nankai University Laboratory Safety Management Measures" (2016) and other Nankai University laboratory safety and security regulations.
3.Experimental safety training: Each participant in the experiment must undergo systematic experimental safety training, including but not limited to laboratory access rules, experimental equipment usage, personal protective equipment (PPE) usage, standardized experimental operations, and emergency handling, etc. In addition, new experimenters need to be approved by the person in charge and other experimental participants and must go through a probationary period under the guidance of experienced experimenters before they can officially participate in the experiment.
4.Standardized operation procedures: Before the formal experiment, we will first determine the standardized operation procedures and conduct small-scale experiments to eliminate potential safety risks. For general experimental operations, we follow widely recognized steps in academia, and consult professors with relevant experience to determine the standard experimental procedures before the formal experiment. For special experimental operations that are not verified or have certain risks involved in the experiment, we will discuss with experts and assess risks, and conduct small-scale experimental verification after careful design. For all experimental steps and data information, we have detailed records for all experimenters to share, ensuring the safety and parallelism of the experiment.
5.Standard equipment and laboratories: We have first and second-level laboratories for conducting different experiments, but mainly in the second-level laboratory, such as cell culture and Salmonella infection, etc. The laboratory is equipped with professional microbiological experimental instruments, as well as biosafety cabinets, chemical fume hoods, and other safety facilities, and safety protection supplies are regularly replaced. Personal items in the laboratory, such as special shoes and experimental clothes, are not mixed. Experimental supplies, equipment, and safety protection supplies are registered and inspected by special personnel every day.
6.Chemical safety: This experiment does not involve chemicals with strong volatility and toxicity, and only a few experiments involve toxic components. Operations with toxic chemicals are all carried out in the fume hood.
7.Biosafety: Although the products of this project do not involve dangerous organisms, there is a certain risk of infection because Salmonella is used to create an infected macrophage model. Therefore, all experiments involving Salmonella are conducted in the biosafety cabinet of the P2 laboratory, and all experimental participants must wear personal protective equipment (PPE) including protective gloves, goggles, N95 masks, experimental clothes, etc., to ensure that no one operates alone and the operator must have more than one month of experience in microbiological operations. The bacteria, cells, and engineered bacteria cultured in this experiment are all cryopreserved or inactivated except for the samples that must be preserved to prevent leakage.
8.Waste disposal: The solid and liquid waste generated by this project is stored separately and processed separately. Solid waste: Disposable equipment, such as pipette tips, culture plates, etc., is treated as waste immediately after use. All waste is sterilized before being handed over to professional biosafety companies for recycling. Reusable equipment, such as conical flasks, beakers, etc., is only generally cleaned if it does not contact microorganisms and hazardous chemicals. If it contacts microorganisms, it is sterilized, and if it contacts hazardous chemicals, it is treated as standard chemical waste. Mouse carcasses are handled by professionals after the experiment. Liquid waste: Liquid waste containing microorganisms or cells is sterilized, and waste liquid containing harmful chemicals is harmlessly treated.
9.Emergency management measures: We have developed a comprehensive response process for different emergency events, including fire, explosion, chemical leakage, and biosafety accidents. Our safety training also covers these contents, and we regularly conduct safety drills to ensure that all experimenters are clear about the response process. In addition, we have arranged safety persons in charge for important links in the experiment, who are responsible for supervising whether the experimental operations are standardized, whether the protective equipment is complete, and whether the experimental reagents are properly stored.
IV.Reducing Environmental Impact
Environmental safety is an important aspect of safety, and our project also considers the potential impact on the ecological environment. We mainly adopt the following methods to reduce potential damage to the ecological environment:
1.Standard laboratories and standardized waste disposal procedures: As mentioned earlier, our laboratories have good sealing and complete facilities, and have standardized and strict waste disposal procedures. There is no experimental biological sample released into the natural environment. Therefore, we have no adverse effects on the natural environment during the research phase of the project.
2.Use of safe biological tools: We chose the globally widespread EcN to make engineered bacteria, which are not invasive and do not express substances that are unfriendly to the environment, and have a small impact on the natural environment. EcN has been widely used in the engineering of microorganisms, and there have been no reports of adverse environmental impacts.
3.Use of natural drug molecules: The main drug molecule, bee venom peptide, is a natural protein derived from bee venom, which is easily decomposed and does not significantly help the survival of the engineered bacteria in the natural environment.
V.User Safety
In the future, our experimental project may be applied to humans. Therefore, we have also considered the safety issues that users of this technology may face. On this topic, we have currently made the following efforts:
1.As mentioned above, the EcN we have chosen does not express toxic factors and has no impact on the human body.
2.We can control the drug concentration within a safe level by controlling the activity of promoters and other means.
In addition, we have also proposed some ideas and made some designs to leave room for future product safety optimization:
1.We have designed a safety plasmid that triggers the engineered bacteria's suicide program when there is no inflammatory signal input.
2.Improve the delivery method of engineered bacteria, such as matching an efficient bacterial delivery system or controlling the engineered bacteria to aggregate in the IBD area, making the engineered bacteria more efficiently targeted to the IBD lesion, reducing the ineffective colonization of engineered bacteria.
3.We can use artificial intelligence and bioinformatics to classify the condition or genetic background of IBD patients and combine clinical data to screen out patients who are most suitable for using this product to reduce ineffective treatment.
VI.Anecdotes about Safety
At the beginning of our project, most team members focused mainly on improving the effectiveness of the product and the innovation of the design, with less attention to safety issues. Under the influence of this atmosphere, we once proposed or tried many radical and even crazy ideas, such as letting engineered bacteria directly express human anti-inflammatory proteins, adding strong promoters to greatly increase the dosage of drugs, etc. However, at a discussion meeting, our instructor immediately rejected these crazy ideas. He said that the human body is a complex system, and too eager to achieve quick success is likely to backfire and cause harm to the human body. He pointed out that our engineered bacteria will ultimately serve patients, and the safety of patients is the top priority. Any responsible technology must put safety issues first, without safety, there is nothing. The instructor also cited the thalidomide incident of the last century to warn us to pay attention to product safety issues. Since then, we have paid more attention to the safety of the product in the design process and put it in the primary position. In the early stage of the experiment, due to lack of experimental experience, we also made some small mistakes on safety issues. In a cell culture experiment, three members of our team accidentally turned on the laboratory's ultraviolet lamp and the lighting lamp at the same time. It took 10 minutes of the experiment before one of the experimenters found and turned off the ultraviolet lamp. After this minor accident, all experimental personnel have developed the habit of checking whether the ultraviolet lamp is turned on after turning on the lights. Of course, we also promptly improved the safety process of the experimental operation, and began to regularly summarize safety issues to ensure that there are no safety hazards in the experimental process. It is in this way that we continuously improve our safety awareness in practice, continuously improve our safety system to improve the overall safety of our project. Because we are well aware that safety issues are not only related to our own safety but also to the safety of patients, nature, and society. Our pursuit of safety reflects our people-oriented values. We always regard the project as a part of promoting individual welfare and serving society.
Safety Plasmid
To ensure the safety of the genetically engineered bacteria in our project, we have designed a safety plasmid to prevent the bacteria from surviving in non-target environments, including natural environments and healthy gut environments. This approach mitigates the risk of unintended consequences, such as colonization in the healthy human gut, which could lead to potential adverse effects, or entering the natural environment and transferring artificial genes to natural bacteria through gene drift. Although we have not yet fully experimentally verified to avoid the leakage of engineered bacteria, we have had a complete design concept for the safety plasmid.
The working principle of the safety plasmid in this project is as follows: under the influence of specific signals (such as inflammatory signals), the safety plasmid remains inactive, allowing the engineered bacteria to survive. Once the signal disappears, the safety plasmid initiates gene expression, thereby triggering the suicide program of the engineered bacteria. To achieve the above functions, the safety plasmid needs to include two main functional elements: sensor and effector.
Sensor
The sensor must possess the capability to identify signals associated with IBD and determine whether the effector works based on the presence or absence of these signal. The key to designing the sensor lies in selecting the appropriate signal and formulating an effective control mechanism.
We have chosen miRNAs that are specifically dysregulated in IBD as the target signal. This miRNA signal exhibits high sensitivity and specificity. It can also directly bind to DNA sequences through base pairing, causing changes in gene structure and thereby altering gene expression. In this project, we use the miRNA that is upregulated by IBD as the target signal molecule.
Figure 1: miRNA
We have designed a gene switch to control the effector. Essentially, this gene switch consists of a miRNA complementary sequence added to the 3'-UTR region of the effector gene. When the effector gene is expressed, the transcribed mRNA's 3'-UTR region contains a complementary sequence to the target miRNA. This complementary sequence can bind to the target miRNA. When the target miRNA is present, the mRNA transcribed by the effector gene binds to the target miRNA through the complementary sequence and is inhibited by it, preventing the expression of suicide-related proteins. Correspondingly, when the target miRNA is absent, gene expression is not blocked, allowing for activation of the effector gene. In this way, the sensor we designed can regulate the activity of the effector by recognizing the miRNA signal related to IBD.
Effector (Effector Gene)
The purpose of the safety plasmid is to make the engineered bacteria commit suicide in non-target areas, so the effector is the suicide gene. When selecting a suicide gene, it is necessary to consider the speed at which the suicide gene takes effect. If the suicide speed is too slow, it may lead to poor effectiveness of the safety plasmid. Conversely, if the suicide speed is too fast, it may make it difficult to introduce the safety plasmid. In screening for suicide genes, we referred to the research results of Huan Liu et al. (2023) and chose the gene corresponding to the CcdB-L42 chimeric protein as the suicide gene. This choice was made because it can inhibit growth after induction in 4 hours and will not be too toxic to prevent introduction.
Figure 2: A and C, Growth curves of strains containing different toxic proteins, CcdB with different split sites and all functional toxic proteins are driven by an inducible promoter. “+” represents induction by arabinose, and “pS8K” is the strain with an empty vector.
Reference: Huan Liu, Lige Zhang, Weiwei Wang, Haiyang Hu, Xingyu Ouyang, Ping Xu, Hongzhi Tang. An Intelligent Synthetic Bacterium for Chronological Toxicant Detection, Biodegradation, and Its Subsequent Suicide. Advanced Science. 2023 Nov 3;10(31):e2304318.