We are committed to designing an advanced engineered bacterium that will specifically target the area of disease in patients with Inflammatory Bowel Disease (IBD) for precise detection and treatment. Given that this engineered bacterium will penetrate deep into the human body and act directly on the diseased area, it is critical to ensure its safety for the human body and the surrounding environment.

In order to achieve this goal, we will take a series of rigorous and comprehensive measures to safeguard the safety of engineering bacteria in practical applications.

Escherichia coli Nissle 1917（EcN） was selected as a chassis microorganism based on its remarkable genotypic properties and excellent safety profile. EcN is a probiotic that is Generally Recognized as Safe (GRAS)[1]. It has inherent antibacterial and anti-inflammatory activity and can be used to treat various intestinal diseases, including inflammatory bowel disease (IBD). EcN belongs to the serotype O6:K5:H1, which has a short LPS side chain, and the K5 pods are serum-sensitive and easily cleared by serum in the body [2] .At the same time, this strain does not secrete any enterotoxin or cytotoxin [1]，and is completely harmless to humans, ensuring its high degree of safety.

In addition, EcN, as an E. coli strain, has a rapid growth cycle and is easy to culture.

Combining these factors, EcN, with its excellent safety and maneuverability, became the preferred chassis microbe for our study.

Photo . Personal protection

Considering the dynamic nature and difficulty of monitoring the levels of disease-related biomarkers, we have incorporated a CRISPR-based genome editing system into the “Entero Guard” engineered bacteria to significantly enhance its ability to amplify inflammatory signals, ensuring that when the biomarker is detected, the drug protein can be secreted into the surrounding environment accurately and effectively.

We selected the TadA-derived Cytosine Base Editor (TadCBE) based on a combination of literature research and expert opinion. To enhance its safety, we added the V106W mutation, which reduces off-target editing, optimizes its editing window and improves C•G-to-T•A selectivity while preserving peak on-target editing efficiency [3]. The TadA-CDd V106W application guarantees the efficacy and precision of probiotic modification.

In this added system, the start codon of the drug protein AvCystatin is replaced by the CBE-editable ACG-tag5 tag sequence, which silences translation of the drug protein, but when the engineered bacterium detects the marker, CBE expressed by PphsA edits the ACG to ATG, which activates translation of the target protein [4] , avoiding the production of drug proteins when they are not necessary.

In addition, we attach great importance to biosafety and environmental protection, and are committed to ensuring the safe use of CRISPR gene editing technology by strictly disposing of all waste generated during the experiment to avoid any unintended impact on the environment or public health.

At this stage, our project does not involve animal experiments. If animal experiments are required at a later stage of the project, we will reassess and improve the safety of our CBE system.

Figure 1. CBE system

Ensuring biosafety is the most important issue for us in our project dedicated to the implantation of engineered bacteria into the human gut. To this end, we have successfully designed a suicide switch mechanism to fulfill the biosafety requirements, including controlling the expression level of the drug in the patient to avoid over-release of the drug and avoiding contamination of the in vitro environment by the engineered bacteria.

In our study, we noticed that: either the patient's condition improved or the engineered bacteria were excreted with fecal matter, there was a decrease in nitric oxide (NO) levels around the engineered bacteria [5] Based on this finding, we utilized the change in NO concentration as a condition to trigger the suicide switch in our design. Once the nitric oxide sensor detects the decrease in NO level, the "suicide switch" in the engineered probiotic will be activated. At this time, the engineered bacteria expressed MazF toxin protein, which triggered programmed cell death by cleaving RNA[6] .This stops the secretion of the drug AvCystatin.

In this way, without the need for additional inducers, the engineered bacteria can initiate a self-destruction mechanism when they detect a decrease in NO levels. This not only avoids the health risks that may be caused by the excessive release of drug proteins, but also ensures the safety and effectiveness of the engineered bacteria in the body. It also ensures that they will not pose a risk to the environment after being excreted from the body, making our design effectively achieve the dual biosafety of engineered bacteria in vivo and in vitro.

In addition, we attach great importance to biosafety and environmental protection, and are committed to ensuring the safe use of CRISPR gene editing technology by strictly disposing of all waste generated during the experiment to avoid any unintended impact on the environment or public health.

In addition, in the suicide system we introduced, MazF, as a toxin protein in the E. coli toxin-antitoxin system, is able to mediate cellular suicide without triggering cell lysis, so there is no need to worry about damaging the original microbiota in the gut.

Figure 2. Suicide system

In the process of participating in the igem competition, our team is well aware that the importance of laboratory safety is no less than that of scientific research and innovation itself. In order to create a safe experimental environment, we strictly abide by the laboratory safety regulations, start from the details, and penetrate the safety awareness into every experimental step.

Basic safety training

Prior to the conduct of the wet experiment, members of the experimental team received comprehensive basic safety training to understand and master basic safety knowledge.

Team members are required to receive comprehensive basic safety training before entering the lab, including chemical safety, biosafety, electrical safety, and personal protective equipment (PPE) use. Our training also covers laboratory rules and regulations, emergency evacuation routes, and first aid measures.

For example, chemical safety: Team members are required to have a thorough understanding of the physical and chemical properties of chemicals commonly found in the laboratory and pay attention to the key information in the Chemical Safety Data Sheets (MSDS), paying special attention to their flammable, explosive, toxic, and corrosive properties, so that proper precautions can be taken when handling them. After understanding their properties, we will strictly classify and store them according to their nature, placing them in designated safety cabinets or storage areas to ensure that they are kept away from sources of ignition, heat and incompatible substances. The storage area should be clearly marked with warning signs for easy identification and management.

Photo 1. Our laboratory reagent storage area
Photo 2. Our safety exits and fire fighter kit

Special skills training

We train our team members in specialized skills, such as microbiological operations and cell culture, according to the needs of the experimental project. We ensure that each member has the ability to complete the experimental tasks independently, and understand the potential risks and preventive measures, and strictly follow the safety code of biological laboratory.

Photo 3. Silhouette of operating technology training

click to Laboratory Safety Management Measures of Medical Laboratory Center of Lanzhou University-Lanzhou University Medical Laboratory Center (lzu.edu.cn)

Personal protective equipment (PPE)

When performing experimental operations, team members must wear appropriate personal protective equipment (PPE), such as lab coats, gloves, goggles, and masks. Also ensure that the PPE is intact during the experiment, and check and replace it regularly.

Photo . Personal protection

division of the experimental area

According to the degree of danger of the experimental objects, the laboratory is divided into different zones, such as low-risk zone, medium-risk zone and high-risk zone. Corresponding safety measures and protective measures are taken in different zones, such as setting up biological safety cabinets and ventilation systems.

Operating Procedures

Before the experiment begins, we develop detailed operating procedures, including steps, safety precautions, and emergency measures. This includes special marking of toxic and harmful reagents used in the experiment to ensure their properties and hazards are understood before use, thereby enabling appropriate protective measures to be taken during the experiment.

For example, when performing nucleic acid gel electrophoresis, the nucleic acid dye EB (Ethidium bromide) used is a strong mutagen with high carcinogenicity. Inhalation, ingestion, or skin contact may cause irritation and injury.

Therefore, the operation must be carried out in a fume hood to avoid inhaling harmful gases, and the operator must wear gloves and goggles to prevent skin contact and eye irritation.

In addition, it is necessary to ensure that the dye in the laboratory effluent must be degraded into non-toxic form.

EB solution purification method:

Add 2.9g of Amberlite XAD-16, a non-ionic polymer adsorbent, to every 100ml of solution; leave it at room temperature for 12 hours ; filter the solution with Whatman 1 filter paper and discard the filtrate; package the filter paper and Amberlite resin in a plastic bag and dispose of it as hazardous waste.

Laboratory Waste Disposal and Classification

We commit that all infectious materials generated during the experiment have been cleared of contamination in the laboratory and treated by high-pressure sterilization, disinfection, etc. before being disposed of.

1) Disposal of Bacterial Waste and Containers Contacting Bacteria

In actual experimental operation, we adopt the following two methods:

----High-pressure sterilization, used for disposing of bacterial waste and glass culture bottles, etc;

----84 Disinfectant Solution Treatment, in which the items to be disinfected are placed in a container containing a chlorine-based disinfectant solution, covered, and sealed. For disinfection of items contaminated with bacterial vegetative cells, use a disinfectant solution containing 2000mg/L of effective chlorine, soak for 2-6 hours or more, disinfect with detergent and running water, rinse, drain, and sterilize by high-pressure sterilization after disinfection.

2) Disinfection of Ultra-clean Bench

Before the experimental operation, clear the ultra-clean bench and disinfect it with ultraviolet light for half an hour; after the operation, clear the ultra-clean bench, wipe it with alcohol, and disinfect the gun with alcohol; then disinfect the ultra-clean bench with ultraviolet light for one hour, and sterilize the consumables used by high-pressure sterilization.

click to Laboratory Safety Management Measures of Medical Laboratory Center of Lanzhou University-Lanzhou University Medical Laboratory Center (lzu.edu.cn)

Photo . Laboratory waste collection container

Other security measures

First of all, before the experiment was officially carried out, we checked the physical and mental health of the team members to ensure that they had the ability and conditions to participate in the experiment, and signed a safety responsibility agreement with each member to clarify their duties and obligations in the laboratory and to enhance their safety awareness and sense of responsibility.

Secondly, we have developed an experiment record sheet to record in detail information such as the time of the experiment, the operator, and the content of the experiment, in order to ensure the standardization and traceability of the experimental process.

In addition, to ensure the cleanliness and tidiness of the laboratory environment, our team has established a detailed schedule for regular cleaning, clarified the specific cleaning areas and responsibilities of team members, and incorporated them into the daily laboratory safety management. A clean lab environment not only significantly improves the accuracy and efficiency of experiments, but also reduces potential safety risks.

Photo 4. Our schedule and the cleaned laboratory

Through these efforts, our team has built a strong defense for laboratory safety and provided a strong guarantee for the smooth progress of scientific research.

Questionnaire privacy security

In order to gain a more comprehensive understanding of the public's as well as patients' perceptions and attitudes toward inflammatory bowel disease (IBD), we distributed questionnaires to the public and patients with IBD, and collected and analyzed the relevant information, with a view to providing data support for improving patient care and public education. We strictly followed the Measures for Ethical Review of Biomedical Research Involving Human Beings issued by the National Health and Wellness Commission of the People's Republic of China in our activities, and we prepared an informed consent form for the general public, which was read by each respondent before filling out the questionnaire, and we ensured that the questionnaire was conducted with the respondents' knowledge and consent. Respondents were given the option to opt out at any time during the questionnaire completion process without having to give a reason. All information will be collected anonymously to ensure that respondents' personal information will not be recorded. In addition, the results of the survey will be kept strictly confidential and all data will be processed anonymously to ensure that respondents cannot be identified. Once the survey is completed, all questionnaire results will be securely stored and only accessible to members of the research team.

Interview privacy security

The LZU-China team conducted personal interviews with the aim of gaining insights into the quality of life of patients with inflammatory bowel disease, their experience of treatment as well as the roles and challenges of healthcare workers in their care, while hoping to gain advice from professionals to optimize the project program. During the interviews, our team pays extra attention to informed consent. All sensitive information in the interviews will be kept strictly confidential and will be used only for the research of this project. Participants may choose to skip any privacy-related questions or terminate the interview at any time, and the right of refusal will be respected. Meanwhile, the interview may be filmed, if you do not wish to appear on camera you can inform the staff, the interview will be recorded in written form. Participation is completely voluntary, and signing the informed consent form indicates understanding of the information and agreement to participate in the study.

Other privacy security

The photos and videos we show on the wiki are taken with the consent and permission of the person themselves, and photos of underage children are taken with the permission of the child and his or her parents.

The engineered strains used in this project are derived from known safe probiotic sources that have been rigorously screened to ensure that they are not pathogenic. The applied CRISPR-Cas9 system has proven its effectiveness in several studies . The product ensures that the therapeutic mechanism is activated at the right time by the precise identification of thiosulfate in the gut, reducing the risk of overdose release. The "suicide switch" mechanism ensures that drug release is terminated when symptoms improve, reducing the potential side effects of continued action and ensuring its safety.

However engineered probiotics may cause an abnormal response in the patient's immune system, leading to allergies or other immune-related side effects. We are prepared to conduct comprehensive allergenicity testing prior to clinical trials and to closely monitor patients' immune responses during the course of the trials. By considering these potential safety issues and their solutions in an integrated manner, it is possible to ensure the safety of engineered probiotic products while providing an effective biologic treatment option for IBD patients. Ongoing research and monitoring will be key to ensuring the successful implementation of this innovative treatment modality.

[1] Sonnenborn,Ulrich,Schulze,et al.The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic.[J].Microbial Ecology in Health & Disease, 2009, 21(3-4):122-158.DOI:10.3109/08910600903444267.

[2] PAN Qiu-Sha, SU Shi-Bing, ZHAO Ming. Advances in functional studies of probiotic Escherichia coli Nissle1917[J]. Microbiology China, 2019, 46(11): 3133-3139 .

[3] Neugebauer ME, Hsu A, Arbab M, Krasnow NA, McElroy AN, Pandey S, Doman JL, Huang TP, Raguram A, Banskota S, Newby GA, Tolar J, Osborn MJ, Liu DR. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat Biotechnol. 2023 May;41(5):673-685. doi: 10.1038 /s41587-022-01533-6. Epub 2022 Nov 10. PMID: 36357719; PMCID: PMC10188366.

[4] Zou ZP, Du Y, Fang TT, Zhou Y, Ye BC. Biomarker-responsive engineered probiotic diagnoses, records, and ameliorates inflammatory bowel disease in mice. Cell Host Microbe. 2023 Feb 8;31(2):199-212.e5. doi: 10.1016/j.chom.2022.12.004. Epub 2022 Dec 27. PMID: 36758520.

[5] Koblan LW, Doman JL, Wilson C, Levy JM, Tay T, Newby GA, Maianti JP, Raguram A, Liu DR. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol. 2018 Oct;36(9):843-846. doi: 10.1038/nbt.4172. Epub 2018 May 29. PMID: 29813047; PMCID. PMC6126947.

[6] Avdagić N, Zaćiragić A, Babić N, Hukić M, Seremet M, Lepara O, Nakaš-Ićindić E. Nitric oxide as a potential biomarker in inflammatory bowel disease. Bosn J Basic Med Sci. 2013 Feb;13(1):5-9. doi: 10.17305/bjbms.2013.2402. PMID: 23448603; PMCID: PMC4333920.