Safety and Security

Introduction

Our team's number one goal in the lab is always safety. We believe it is essential to prioritize the health of our team, our university community, and the environment. Each year, we incorporate safety into our project design and our lab work.

iGEM Rules and Policies

• Our team is not completing any of the activities prohibited by iGEM such as using organisms from Risk Group 3 or 5, using parts from an Organism in Risk Group 4, releasing or deploying a genetically modified organism, or testing our product on humans.

Lab Safety

• The GSU-SWJTU iGEM team works in a Biosafety Level 2 (BSL-2) laboratory. Therefore, our biosafety protocol is modeled after the Georgia State University biosafety protocol. Lab access is strictly limited through card access to members of the iGEM team and other trained university researchers. Before working in the lab, all team members received safety training, including security awareness, right-to-know, hazardous waste, lab safety, and autoclave training. In the lab, food and drink are forbidden (pizza parties strictly kept in the hallway), and personal protective equipment (PPE) is worn at all times, including gloves, and goggles as necessary. Before exiting the lab, all workspaces are sterilized with ethanol or bleach, and students decontaminate via handwashing. With these measures, we comply with the protocol of our institution as well as making sure all lab members do not carry any lab risks outside.

Managing Overall Risk

• To manage risks in our project, all team members were required to complete institutionally mandated laboratory safety training modules, including Laboratory Safety Training, Autoclave Safety, Biosafety Cabinet Usage, and Hazardous Material Disposal. Completion of these trainings is a prerequisite for obtaining lab access, ensuring that only trained personnel can enter and work in designated spaces. Our laboratory work is conducted in a BSL-1 or BSL-2 compliant facility equipped with proper containment equipment such as biosafety cabinets and autoclaves. We follow institutional protocols for waste inactivation and disposal, including autoclaving biological materials before disposal and using designated waste streams for hazardous chemicals. Furthermore, to reduce potential risks of environmental release, our project design incorporates biocontainment strategies. For example, we limit the use of antibiotic resistance markers. These precautions, along with strict adherence to university biosafety guidelines, ensure comprehensive risk mitigation throughout the experimental process.

Safe Project Design

• Safety is at the heart of our project design. We intentionally selected two well-studied and non-pathogenic chassis, Escherichia coli and Lactobacillus, for DNA insertion. The E. coli strains we use (such as K-12 derivatives) are classified as non-pathogenic and safe for laboratory use. Our chosen Lactobacillus species is also well characterized and recognized as safe. All genetic parts designed in our project are non-harmful to humans, animals, or plants. Although we also work with Streptococcus mutans, a bacterium associated with dental caries, all work involving this organism is conducted under appropriate biosafety level 2 (BSL-2) conditions, following institutional and iGEM safety guidelines.

Killswitch Systems

With biosafety and security at the forefront of our minds, our team set out to develop an induced Toxin Antitoxin system for our bacteria we plan to introduce to kill S. mutans. The goal of this system is to control lactobacillus, ensuring that they cannot survive outside our target environments.

• Our killswitch is based on the TA (Toxin-antitoxin) system and an L-Asp sensor. The TA system, composed of a toxin gene and an antitoxin, the latter counteracting the toxicity of the former (Fraikin et al., 2020). TA systems are linked to many biological processes, such as the formation of antibiotic-tolerant cells and the stabilization of mobile elements (Van Melderen & Saavedra De Bast, 2009). Based on its functions, TA systems are divided into 8 categories, and the MazEF, which we are using, belongs to type II, meaning that MazE (Figure 1) antitoxin directly binds to MazF (Figure 2) toxin and forms a protein-protein complex, resulting in neutralization (Dai et al., 2021). After MazE degradation, MazF is released from the complex and acts as an endonuclease that cleaves RNA.

  

• For the switch of this TA system, we plan to use an L-Asp sensor. In Acinetobacter baylyi ADP1, AalR is a LysR-type transcriptional regulator that specifically responds to L-Asp. When L-Asp is present, it binds to AalR, causing a conformational change that enables AalR (likely as a tetramer) to bind to a conserved operator motif (ATGC-N₇-GCAT) upstream of the aspA gene. This binding activates transcription of aspA, which encodes aspartate ammonia-lyase, as well as transport- and racemase-related operons (racD-aspT). Without L-aspartate, AalR still binds to the promoter region but in a conformation that blocks RNA polymerase recruitment. This results in very low or basal expression of the target genes, keeping the system repressed until the presence of the inducer. Loss of aalR abolishes L-Asp utilization, confirming its essential role. Overall, AalR acts as a key sensor and transcriptional activator that links environmental L-Asp availability to metabolic gene expression (Bedore et al., n.d.). Based on this information, we came up with our L-Asp sensor. When there is L-Asp, which will be introduced to the oral condition by the toothpaste if the original concentration of it is not enough to kick on the kill switch, AalR will activate the production of MazE by binding to the consensus region in the aspA promoter, which will be used to neutralize MazF. When there is no L-Asp or its concentration is below the threshold, the transcription of MazE will be inhibited, and MazF will cause cell death. To control the cytotoxicity, we used a T7 promoter to control the initiation of mazF. The T7 promoter is a strong promoter that specifically requires T7 RNA polymerase to initiate transcription. In E. coli BL21(DE3) and similar strains, the gene encoding T7 RNA polymerase is integrated into the chromosome under the control of the lacUV5 promoter (a variant of the lac promoter). The lacUV5 promoter is regulated by the LacI repressor. When isopropyl-β-d-thiogalactoside (IPTG) is added, it binds to LacI, releasing its repression. This allows transcription of the t7rnap gene, leading to the production of T7 RNA polymerase. The polymerase then recognizes the T7 promoter on a plasmid and drives strong expression of the target gene. As we will test our system in E.coli first, adjusting the concentration of IPTG will be a good control. When we transport this system into Limosilactobacillus reuteri in the future, as there is no IPTG transporter in L. reuteri, we will replace this promoter with another constitutive promoter. However, after we conducted the alignment of aalR in ADP1 with E.coli and L. reuteri, we found high similarity with high randomness and low similarity, respectively, indicating that there is no similar AalR that has a similar function in these two strains. Therefore, we need a plasmid to express AalR. As AalR is the regulator protein, we used the constitutive promoter p32 to carry out its transcription. To avoid read-through between parts, we introduced a strong double terminator (BBa_B0015) consisting of BBa_B0010 and BBa_B0012 (Figure 3) for mazE and aalR. For mazF, cause there is terminator initially in the backbone with the enough strength, there is no need to introduce a new terminator. Because the TA system is very coordinated, we need to adjust the transcriptional strength of toxin and antitoxin, which means to avoid the overproduction of toxin to kill the cell too early, but at the same time ensure enough toxin to kill L. reuteri when there is no S. mutans. Therefore, the strength of ribosome binding site (RBS) should be taken into consideration. After careful consideration, BBa_B0034, with a comparative strength of 0.38, is for the antitoxin, and BBa_B0033, with 0.0133, is for the toxin. For AalR expression, we use BBa_B0032, with 0.047, to ensure that this expression will not cause too much burden for the host. The best way to adjust the TA system is to build an RBS library with different strengths of RBS, but we are limited by time and financial constraints to do this. We have suggested a draft for it instead.

  

• The pUPD3 plasmid was employed as the backbone for expression of the AalR regulator protein and confers chloramphenicol resistance. The pET-21(+) vector was selected for expression of MazF, as it additionally harbors the LacI repressor and provides resistance to ampicillin. The pT7 plasmid was utilized for expression of MazE, which carries kanamycin resistance; notably, the absence of an endogenous RBS and transcription terminator in this construct facilitates the incorporation of customized regulatory elements. Together, these vectors were chosen to ensure compatibility of resistance markers, allow precise regulation of TA expression, and provide flexibility in genetic circuit design.

S. mutans

• Streptococcus mutans is the target bacterium for this whole project, in which we are designing L. reuteri to produce nisin to kill S. mutans. We will be running inhibition assays with S. mutans to check the success of the L. reuteri at killing S. mutans as well as checking biofilm disruptions.

Risks of S. mutans

• Handling Streptococcus mutans in the lab poses risks if cultures are accidentally aerosolized or come into direct contact with skin or open wounds, potentially leading to infections and causing inflammation of heart valves. A subset of strains has been linked to other extraoral pathologies such as cerebral microbleeds, IgA nephropathy, and atherosclerosis. Handling Streptococcus mutans also requires caution due to its role in dental decay, which requires proper biosafety procedures. The risk given by the provider of S. mutans was BSL 1 based on its risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL) by the U.S. Department of Health and Human Services.

Measures in Place to Manage the Risks Identified

• All work is conducted under BSL-2 practices, with no procedures performed on open benches if there is a risk of aerosol generation. Waste is autoclaved prior to disposal, and work surfaces are disinfected with ethanol after use. Direct contact with the mouth and skin is strictly avoided. To ensure safe practices, all team members are required to complete institutionally mandated laboratory safety training—including laboratory safety, autoclave safety, biosafety cabinet usage, and hazardous material disposal—before gaining lab access. Furthermore, S. mutans are kept in its own part of the lab with its own incubators. We also only let experienced students handle the S. mutans to decrease the likelihood of accidental exposure. These measures, combined with strict adherence to university biosafety guidelines, provide comprehensive risk mitigation throughout the experimental process.

Summary

By identifying biological, chemical, physical, and security risks, and implementing targeted mitigations, we ensured that our project was conducted responsibly. Safety was not an afterthought but a guiding principle throughout the engineering cycle.

References

• Bedore, S. R., Schmidt, A. L., Slarks, L. E., Duscent-Maitland, C. V., Elliott, K. T., Andresen, S., Costa, F. G., Weerth, R. S., Tumen-Velasquez, M. P., Nilsen, L. N., Dean, C. E., Karls, A. C., Hoover, T. R., & Neidle, E. L. (n.d.). Regulation of l- and d-aspartate transport and metabolism in acinetobacter baylyi ADP1. Applied and Environmental Microbiology, 88(15), e00883-22. https://doi.org/10.1128/aem.00883-22
• Biological Safety - Georgia State University Research Services & Administration. (2025). University Research Services & Administration website: https://ursa.research.gsu.edu/research-safety/biosafety/
• Dai, J., Chen, Z., Hou, J., Wang, Y., Guo, M., Cao, J., Wang, L., Xu, H., Tian, B., & Zhao, Y. (2021). MazEF toxin-antitoxin system-mediated DNA damage stress response in deinococcus radiodurans. Frontiers in Genetics, 12. https://doi.org/10.3389/fgene.2021.632423
• Fraikin, N., Goormaghtigh, F., & Van Melderen, L. (2020). Type II toxin-antitoxin systems: Evolution and revolutions. Journal of Bacteriology, 202(7), e00763-19. https://doi.org/10.1128/JB.00763-19
• Lemos, J. A., Palmer, S. R., Zeng, L., Wen, Z. T., Kajfasz, J. K., Freires, I. A., Abranches, J., & Brady, L. J. (2019). The Biology of Streptococcus mutans. Microbiology Spectrum, 7(1). https://doi.org/10.1128/microbiolspec.gpp3-0051-2018
• Shetty, R. (2003, July 17). Part:BBa B0015 - parts.igem.org. Parts.igem.org. https://parts.igem.org/Part:BBa_B0015
• Streptococcus mutans Clarke - 25175 | ATCC. (n.d.). Retrieved from www.atcc.org website: https://www.atcc.org/products/25175
• Van Melderen, L., & Saavedra De Bast, M. (2009). Bacterial toxin-antitoxin systems: More than selfish entities. PLOS Genetics, 5(3), e1000437. https://doi.org/10.1371/journal.pgen.1000437
• UniProt. (2025). https://www.uniprot.org/uniprotkb/P0AE72/feature-viewer#structure