We have analyzed suicide gene switches from the past six iGEM teams, all of which focused on the field of gut microbiota, similar to our team's focus. We've identified a common challenge among these teams - the inability to induce complete gene suicide effectively. The most successful approach among them was a low-temperature-induced suicide system, but it posed constraints on maintaining a constant temperature of 37°C throughout the project, as the bacterial strain would die at lower temperatures.

Furthermore, some recent articles have explored using high-temperature induction to cause bacterial death. However, these methods often involve complex techniques like Focused Ultrasound (FUS) to generate heat ex vivo to induce engineered bacterial death.

Given that colorectal cancer typically occurs in proximity to the rectum, we have introduced an innovative concept of using suppositories. We have also replaced the complex Focused Ultrasound (FUS) heating system with a more cost-effective and efficient heating suppository approach.

In consideration of potential biosafety concerns associated with our project, we have designed a suicide system.

In the suicide system, we have selected pTcl42 as the promoter, which allows the engineered strain to express MazF. After the engineered strain has completed its therapeutic role, patients can introduce a heating suppository. When it is necessary to completely eliminate the engineered strain, the suppository activates its heating function to trigger the bacterial suicide system.
Temperature-Controlled Promoter Tcl42Temperature-inducible promoters are a class of promoter sequences that regulate gene transcription activity within specific temperature ranges. They are widely used in the fields of synthetic biology and genetic engineering to achieve temperature-sensitive gene expression control.The mechanism of temperature-controlled promoters relies on temperature fluctuations to alter promoter activity, thereby regulating gene transcription levels. At low temperatures, these promoters are inhibited, resulting in low transcriptional activity. In contrast, at higher temperatures, they become activated, leading to increased gene transcription. This temperature sensitivity is achieved through the interaction of specific regulatory elements within the promoter sequence and temperature-sensing factors.We have chosen the pTcl42 promoter because it has an ideal activation temperature threshold of 42°C, with low expression levels at 37°C. In its natural host, Salmonella enterica serovar Typhimurium, Tlp is presumed to regulate virulence genes upon entry into a warm host organism. Additionally, Tcl is a temperature-sensitive mutant of the bacteriophage lambda's "cl" gene. In its natural environment, cl serves as a transcriptional repressor, allowing the phage to establish and maintain latency.

Mechanism of Action of MazFMazF belongs to the toxin-antitoxin (TA) systems, which are extensively studied and have well-defined mechanisms. The mazF gene is downstream of the TA system mazEF, encoding a stable toxin protein. MazF is an mRNA interferase (ribonuclease) that acts independently of ribosomes. It can specifically cleave single-stranded mRNA at certain sequence sites and is highly conserved in most microorganisms and some archaea.In Escherichia coli, MazF recognizes ACA sequences and hydrolyzes the phosphodiester bond at the first A position of either the 5' or 3' end, leading to ribosome release from cleaved mRNA and the inhibition of protein synthesis. Consequently, aberrantly encoded polypeptides are released and degraded by intracellular proteases, ultimately resulting in cell death.

The basal transcription activity of the gene can be regulated within a specific temperature range by using a temperature-controlled promoter. We chose the pTcl42 promoter because its ideal activation temperature threshold is 42°C, and it has low expression levels at 37°C. MazF is a widely studied toxin-antitoxin (TA) system in Escherichia coli, and its mechanism of action is well defined. MazF can recognize ACA sequences and hydrolyze the phosphodiester bond at the first A position at either the 5' or 3' end, causing ribosome release from cleaved mRNA and preventing protein synthesis. Subsequently, improperly encoded polypeptides are released and degraded by intracellular proteases, leading to cell death. Therefore, we use the temperature-controlled promoter TC1-42 and mazF to induce bacterial lysis. In order to achieve temperature-induced self-lysis in the engineered strain, the pTcl42 promoter was placed upstream of the bacterial lysis gene mazF. Subsequently, the Tcl42 promoter and the mazF gene were cloned together into the pSB1A3 plasmid.

The successfully constructed plasmid was transformed into E. coli DH5a bacteria using heat shock method, and recombinant bacteria were screened on LB agar plates containing 100 μg/mL ampicillin. To evaluate the expression of the mazF gene at different temperatures, the transformed bacteria were cultured for 12 hours at 37°C and 42°C, and their OD600 values were measured using a spectrophotometer. Wild-type DH5a and DH5a carrying only the pTcl42 of pSB1A3 plasmid were used as controls. All experiments were performed in triplicate to ensure the reliability of the results. The results are shown in Figure 2A.
To evaluate the time dependence of the bacterial lysis system driven by the pTcl42 promoter at 42°C, a time-course test was performed. The engineered bacteria were added to a 96-well plate and cultured in a shaking incubator at 42°C. Every 2 hours, the OD600 values were measured using a microplate reader to assess bacterial growth. The results are shown in Figure 2B. All experiments were performed in triplicate to ensure the reliability of the results.
The experimental results showed that the temperature-inducible promoter was induced at 42°C, causing almost complete lysis of the bacteria within 12 hours. In the future, we plan to modify common suppositories to have a heating function. When it is necessary to completely kill the engineered bacteria, the heating function of the suppository will be activated to initiate the bacterial suicide system.

Almost all projects related to gut microbiota have traditionally relied on oral administration. However, oral administration of synthetic biology-based interventions for gut microbiota presents challenges, including gastrointestinal discomfort such as diarrhea and bloating, as well as potential risks of infections or overgrowth associated with engineered microorganisms. While these interventions involve the deliberate manipulation of microbial populations in the digestive tract to achieve specific health benefits or therapeutic outcomes, careful consideration of safety and potential side effects is crucial in their development and implementation.

In addition, we conducted a thorough review of previous iGEM team projects and found that none of them had utilized suppositories as a delivery method. Nevertheless, after in-depth research, we unanimously believe that, for colorectal cancer-related projects, using suppositories offers distinct advantages over oral administration. To support this notion, we consulted medical professionals who provided a highly favorable assessment from a clinical perspective.

A synthetic biology project centered around gut microbiota entails the engineering of microorganisms to enhance or modify their functions within the gastrointestinal tract for therapeutic or health-related purposes. Transitioning from oral administration to a suppository (rectal) delivery method presents several potential benefits. It may enhance the stability and viability of engineered microorganisms in the harsh acidic stomach environment, improve targeted delivery to the lower digestive tract, and reduce exposure to digestive enzymes. Consequently, this transition can increase the intervention's effectiveness while minimizing potential side effects and discomfort often associated with oral ingestion.

In order to promote the use of suppositories in the field of gut synthetic biology, our team has created a guidance manual tailored to this direction. Future teams can refer to this manual to better integrate their projects with the use of suppositories.
This user manual aims to provide detailed guidance on how to use suppositories in gut microbiota synthetic biology projects. We will delve into why suppositories are chosen as a delivery method and how to effectively implement this approach. The manual covers various aspects of the project, including safety considerations, laboratory techniques, data collection and analysis, as well as team collaboration.

Utilizing suppositories in gut microbiota projects represents an innovative approach with significant scientific and practical implications:Safety and Effectiveness: This manual emphasizes the advantages of suppositories as a delivery method, reducing potential side effects compared to oral administration, enhancing therapeutic efficacy, and minimizing disruption to the natural gut microbiota.Scientific Innovation: Pioneering the use of suppositories in gut microbiota projects advances the fields of synthetic biology and microbiome research, offering a new avenue for addressing complex health issues.Safety and Compliance: By addressing safety considerations and ethical regulations, this manual ensures project safety, compliance with iGEM competition rules, and upholds high standards of laboratory and clinical practices.Team Collaboration: Project success hinges on seamless teamwork, and this manual provides clear role definitions and responsibilities, facilitating the smooth execution of projects as planned.In summary, this user manual is designed to serve as a comprehensive guide for future iGEM teams, assisting them in implementing gut microbiota synthetic biology projects and achieving breakthroughs in this field while ensuring safety, compliance, and scientific innovation.

In addition to achieving our experimental goals and obtaining the desired target genes, many of our team members actively participated in local scientific seminars. These seminars provided a platform for engaging in discussions and knowledge exchange with other iGEM university teams and professionals in our project-related industries. This collaborative effort aimed to refine our project design and experimental processes, striving to make our product as efficient and safe as possible.

In July of this year, our team was invited by the China iGEM Competition Committee to attend the largest academic symposium in the China iGEM region, held in Hainan. During the symposium, we attentively listened to project presentations from other university teams. The diverse project directions and imaginative ideas shared by these teams greatly benefited our own team. One common trait among these teams was their strong emphasis on and vigilance regarding biosafety. Their innovative approaches to handling various aspects of the experimental process opened our eyes to new possibilities.

As we continued to share insights during the symposium, our team continually refined our design plans and addressed safety concerns that had previously been overlooked. Following the event, our team engaged in private discussions with two other teams from the fields of pharmacy and biology in Beijing. These university teams had research directions similar to ours. Their wealth of university experience and laboratory expertise provided our team with valuable safety case studies, expanding not only the depth of our biological knowledge but also broadening our overall understanding. These interactions laid the foundation for our subsequent successful and safe experiments.
Subsequently, on the eve of the National Day holiday, representatives from our team participated in a collaborative biosafety awareness exhibition organized by multiple university iGEM teams from across the country. While our project's specific objectives differed from those of other teams, their wealth of experience and expertise still contributed to the improvement and enhanced safety of our project design.

Communicate with the team representatives of CUT-China

Group photo after communication with XJTLU-China team representatives

Listen to the UST-China team's project sharing

Before entering the laboratory, our team meticulously reviewed safety guidelines from various laboratory resources. Under the guidance and supervision of the three faculty members forming the Biological Safety Oversight Committee, we underwent on-site safety training. Throughout the experimental process, we consistently held ourselves to high standards of biosafety. Prior to conducting each experiment, we thoroughly assessed its safety, engaging in discussions within the team and with our mentors. We only proceeded with experiments after ensuring their safety and stability.
During experiments, we maintained the laboratory's integrity and conducted disinfection checks for personnel entering and exiting, ensuring that all genetic engineering experiments remained confined to the laboratory and preventing any potential gene spillage. Our approach to safety extended from the initial gene concept, through the laboratory doors, and further as we exited the laboratory.