In laboratory work, the correct and safe use of instruments and equipment is a key prerequisite for ensuring smooth progress in research. Before launching our project, we conducted systematic safety training on commonly used equipment, covering heat sources and heating devices, aseptic operation equipment, centrifuges and shakers, molecular biology instruments, as well as analytical and culture equipment. During the training, we learned that when using heat sources, it is essential to prevent alcohol evaporation and splashing, and to avoid adding fuel while the flame is lit; when operating laminar flow hoods and biosafety cabinets, we must maintain a sterile environment and prevent airflow disturbance; centrifuges and shakers require proper sample balancing and stable equipment placement; electrophoresis systems, PCR machines, and other molecular biology instruments demand attention to electrical safety, along with the use of protective equipment when working with hazardous dyes or ultraviolet light sources; meanwhile, balances and incubators must be operated with attention to stability, sealing, and proper waste disposal. Through systematic learning and strict adherence to these guidelines, we have gradually developed the principle of “prevention first, adequate protection, and standardized operation” in our daily experiments, which not only ensures the safety of laboratory personnel but also lays a solid foundation for the reliability of our research outcomes.
In terms of personal safety protection, all team members received targeted guidance and training before entering the laboratory. Upon entry, they must wear long-sleeved lab coats and long pants, put on gloves, and tie back long hair, ensuring that lab coats are fully buttoned. The use of cosmetics, jewelry, or contact lenses is prohibited, as is keeping long fingernails. During experiments, lab coats must not be unbuttoned or sleeves rolled up. When leaving the laboratory, protective gear should be removed, and used gloves must be properly disposed of. Laboratory conduct must follow strict regulations: eating and drinking are strictly forbidden, as is touching the mouth, nose, or eyes with hands. Personal belongings and lab notebooks should be placed in designated areas, and gloves must be removed before using phones or other devices. By adhering to these personal protection requirements, team members can effectively reduce the risk of accidents and cross-contamination during experiments, ensuring their own safety while maintaining the cleanliness and hygiene of the laboratory environment.
In terms of strain safety, we mainly used three Escherichia coli strains: BL21, DH5α, and Nissle 1917, all of which belong to Biosafety Level 1 (BSL-1).
E. coli BL21 is a commonly used protein expression strain in laboratories, classified as BSL-1. This strain lacks multiple exotoxin genes, can efficiently express heterologous proteins, and is widely applied in molecular biology and protein engineering experiments. BL21 poses minimal environmental risk and has high operational safety; routine gloves and aseptic techniques are sufficient for handling. With long-term laboratory use and passaging, BL21 remains genetically stable and is suitable for safe protein expression and related research under standard experimental conditions [1].
E. coli DH5α is widely used for DNA cloning and plasmid amplification, also classified as BSL-1, requiring no advanced biosafety precautions. This strain has been genetically modified to lack pathogenic factors, making it well-suited for DNA amplification and plasmid construction in molecular cloning. DH5α has minimal environmental impact, high operational safety, and stable traits after engineering, making it one of the most common safe laboratory strains in molecular biology and providing a reliable experimental foundation for DNA manipulation in our project [1].
E. coli Nissle 1917 is a probiotic strain classified as BSL-1, requiring only standard gloves and aseptic procedures for safe handling. It lacks Shiga toxins, enterotoxins, pathogenicity islands, and virulence factors. Its long-term clinical application (e.g., in the drug Mutaflor) has shown no cases of infection or toxic side effects, and it has been officially recognized by regulatory authorities in Germany, Canada, and several European countries. Nissle 1917 exhibits strong resistance to gastric acid, bile salts, and oxidative stress, allowing it to stably colonize complex physiological environments. It is particularly suitable for oral delivery systems and as a chassis for drug or vaccine vectors. Additionally, it possesses unique adhesins and intestinal colonization factors, enabling long-term persistence in the host gut. Engineered Nissle 1917 can be coupled with biocontainment strategies (e.g., suicide switches), demonstrating high stability and environmental safety, making it an excellent experimental chassis strain [2].
Proper disposal of laboratory waste is a crucial step in ensuring both personnel safety and environmental protection. According to the nature of the waste, it is generally classified into four categories: non-infectious, infectious, sharps, and chemical waste. Non-infectious waste, such as uncontaminated packaging materials, can be directly disposed of in regular trash bins for municipal processing. Infectious waste, including used disposable gloves, pipette tips, and bacterial culture preservation solutions, must be sterilized via autoclaving or other disinfection methods before being handled according to medical waste regulations and disposed of by specialized companies. Certain glassware that can withstand high temperatures, such as test tubes, Petri dishes, and conical flasks, can be sterilized and reused. Sharps waste, such as needles, blades, and broken glass, whether contaminated or not, must be collected in dedicated sharps containers. When the container is filled to about three-quarters, it should be sealed and sent to a licensed disposal facility. Chemical waste, including toxic, corrosive, flammable, or explosive liquids and organic solvents, must be collected separately according to their chemical properties in compatible, labeled containers. Labels should indicate the main components, concentration, hazards, date, and responsible person. Disposal must be carried out by qualified units following safety protocols. Strict adherence to these waste management practices minimizes experimental risks and ensures the safety of laboratory personnel and the environment.
Design of Safety Systems
In our engineered bacterial project, the design of the safety system is a central and critical component. Ensuring both laboratory operational safety and the controllability of engineered bacteria in human and space environments relies on robust safety mechanisms. We have carefully considered potential risks of genetic leakage, including scenarios where engineered bacteria may escape into the external environment or remain in the gut.
To address the latter, we designed an arabinose-inducible in vivo suicide system, enabling engineered bacteria to self-clear within the intestinal tract. For external disposal, our research and analysis indicate that once the bacteria are excreted, no additional complex treatment is required; simple collection followed by high-temperature incineration is sufficient to completely inactivate them.
Through this multi-layered protection and strictly controllable design, we ensure that the engineered bacteria stably express target products within controlled conditions while allowing rapid clearance when necessary, thereby minimizing potential risks to the greatest extent.
Design of the Arabinose-Inducible Suicide System
The arabinose operon is a bacterial gene regulatory system that suppresses the transcription of associated genes in the absence of L-arabinose. When L-arabinose is present in the environment, it induces a conformational change in the AraC protein, relieving its repression of the pBAD promoter and thereby activating transcription of downstream genes [3]. This mechanism allows bacteria to rapidly initiate metabolic pathways when arabinose is available, effectively regulating their metabolic activity.
Figure 2. Design of the Arabinose-Inducible Suicide System
In our system, the incorporation of the arabinose operon provides a safety mechanism for patients. If adverse effects occur, or if the secretion of HMB or serotonin by the probiotic is no longer needed, patients can administer arabinose to induce bacterial expression of lytic enzymes, triggering self-clearance. This ensures that the probiotics remain under controllable conditions at all times, reducing potential risks associated with their use.
Validation of the Arabinose-Inducible Suicide System
Figure 3. Experimental Design for Validation of the Arabinose Promoter
First, we constructed an arabinose promoter testing system using red fluorescent protein (RFP) as a reporter to assess its expression under different concentrations of arabinose induction.
Figure 4. Validation of the Arabinose Promoter
Next, we measured the fluorescence intensity (excitation wavelength: 584 nm; emission wavelength: 607 nm) and OD600 values. The normalized fluorescence ratio (Fluorescence/OD600) was then calculated. The results demonstrated that as the concentration of arabinose increased, the fluorescence-to-OD600 ratio steadily rose, indicating that the arabinose operon can effectively induce the expression of the corresponding protein in response to arabinose.
Testing of the Arabinose-Inducible Suicide System
Figure 5. Validation of the Suicide System
We obtained the MazF sequence derived from Escherichia coli K12 from the NCBI database and amplified it via PCR. Subsequently, we genetically engineered the arabinose operon pBAD with the MazF sequence and evaluated bacterial survival under arabinose induction. The results showed that after adding 0.5% L-arabinose to the bacterial culture at 5 hours, the OD600 of the culture began to decline significantly and reached zero by the 25th hour.
Figure 6. Waste Management in Space
A. Toilet in Space B. Sealed Bags for Space Excreta [4–5]
According to our team’s investigation, in the space station, astronauts’ excreta undergo strict collection and treatment. Urine is processed through the Water Recovery and Recycling System, where it is purified and reused; after multi-stage filtration and distillation, it is converted back into drinking water to meet astronauts’ daily needs. Solid feces are sealed in specially designed bags containing preservatives and deodorizing agents. These bags are impermeable, vacuum-resistant, and resistant to microbial degradation, allowing them to remain stable during long-term missions. After sealing, the feces undergo additional treatments such as vacuuming and UV/chemical sterilization to further reduce biological risks, and are ultimately stored in the waste compartment [4].
During space station resupply or mission completion, these sealed waste packages are loaded onto cargo spacecraft. After completing their mission, the spacecraft are deliberately deorbited to re-enter Earth’s atmosphere. During re-entry, the extreme friction with air generates temperatures of several thousand degrees Celsius, causing all materials to rapidly vaporize and incinerate completely. This process ensures that any engineered bacteria in the feces cannot survive or spread into the natural environment [5].
Compared to terrestrial laboratories or medical settings, space features a far stricter closed-loop management system. On Earth, improper disposal of laboratory waste still carries a theoretical risk of leakage. In contrast, on the space station, all excreta and waste strictly follow the procedure of “full collection → sealing → vacuum sterilization → centralized storage → high-temperature incineration,” forming a physically irreversible ultimate safety barrier. This not only eliminates the possibility of engineered bacteria freely spreading in space but also prevents any potential ecological risks when returning to Earth (Fig.7) [6].
Figure 7. Workflow for Space Excreta Management
In summary, even if engineered bacteria are excreted along with feces, they will be completely eliminated through the dual safeguard mechanisms of waste processing and incineration during atmospheric re-entry, thereby achieving the highest level of safety for external containment.
Summary
Through systematic safety training, strict laboratory protocols, careful strain selection, and multi-layered safety system design, this project has achieved high standards of biosafety both inside and outside the laboratory. Whether in terrestrial experimental settings or the unique conditions of space, we have established a safety strategy based on “prevention first, standardized operation, and controllable clearance,” ensuring that research on engineered bacteria is conducted safely, compliantly, and responsibly. This approach not only protects laboratory personnel and the environment but also lays a solid safety foundation for the application of synthetic biology outcomes.
Reference
[1]. Chart, H., Smith, H. R., La Ragione, R. M., & Woodward, M. J. (2000). An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha and EQ1. Journal of applied microbiology, 89(6), 1048–1058. https://doi.org/10.1046/j.1365-2672.2000.01211.x
[2]. Dubbert, S., Klinkert, B., Schimiczek, M., Wassenaar, T. M., & Bünau, R. V. (2020). No Genotoxicity Is Detectable for Escherichia coli Strain Nissle 1917 by Standard In Vitro and In Vivo Tests. European journal of microbiology & immunology, 10(1), 11–19. https://doi.org/10.1556/1886.2019.00025
[3]. Guzman, L. M., Belin, D., Carson, M. J., & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of bacteriology, 177(14), 4121–4130. https://doi.org/10.1128/jb.177.14.4121-4130.1995
[4]. https://baijiahao.baidu.com/s?id=1831176080440570945&wfr=spider&for=pc
[5]. https://baijiahao.baidu.com/s?id=1828200119887822952&wfr=spider&for=pc
[6]. https://baijiahao.baidu.com/s?id=1814787152482518696&wfr=spider&for=pc
