Overview

For various usage scenarios of Luminaid, such as in ocean and space environments, it's important to analyze the specific environment of each scenario to identify risk factors. This analysis will help us design and refine safety modules to ensure the proper functioning of Luminaid and the safety of users in different scenarios.

Comparative dimension	Ocean	Space	Conclusion
Temperature	Hypothermia (susceptible to hypothermia)	The average temperature of space is -270.3 °C, with different surface temperatures on different celestial bodies (e.g., the Moon's extreme low temperature can reach -180 °C on lunar nights and 150 °C on lunar days).	In the ocean and space: the need to choose insulation materials
Atmosphere	Earth's atmosphere	High vacuum environment (inhospitable)	In space: the need for life support systems
Radiation	Radiation in partially contaminated sea areas	Strong radiation environments (ionizing radiation at the core)	In space: the need to protect against ionizing radiation
Microbial infections	Gram-negative bacteria are the main pathogenic microorganisms that can easily cause infection through wounds	Complexity of microorganisms in space; microgravity can cause a decrease in the functioning of the astronaut's immune system	In the ocean and space: the need to protect against microbial infections

To see more details of the Comparison of Ocean and Space Environments, please click here to go to the corresponding section of the HP page.

Different environments have different safety requirements. We have compared various environments and focused on key aspects to make design improvements. However, the high complexity of environments, such as the ocean or space, poses a significant challenge for our program. Further efforts will be needed to enhance safety in the future.

Estimating the toxicity of genetic circuits to chassis cell

According to the definition provided by the European Committee for Standardization (CEN) in its Document No. 30 of 1992, cytotoxicity refers to cell death, cell lysis, and inhibition of cell growth caused by products, materials, and their leachates^[3]. There exists a close interaction between the chassis cells and their loaded genetic circuits. Overexpression of genetic elements can trigger cellular growth stress^[4], while specific regulation of these elements can also lead to cytotoxicity. For genetic circuits, when chassis cells experience growth retardation due to growth stress or cytotoxicity, it adversely affects the predictability and genetic stability of the artificially constructed genetic circuits within them.

Currently, the assessment of cytotoxicity often involves investigating two aspects: cell growth rate and survival rate^[5]. However, there is a lack of a unified consensus providing direct reference for determining the weights of these indicators. We have employed the Analytic Hierarchy Process (AHP) to comprehensively determine the weight values based on the consultation results from three experts:

where wg represents the weight of growth rate, while ws stands for the weight of survival rate.

Growth rate serves as a direct indicator of cellular proliferation capacity. For many cell types, a decrease in growth rate is an early and sensitive sign of cellular damage or toxicity. However, survival rate is a crucial metric for assessing cellular health status and toxicity, as it directly reflects the ability of cells to maintain vital activities after exposure to genetic circuits. Given its paramount importance over growth rate, survival rate is assigned a higher weight.

To quantify the relative value of cytotoxicity, the percentage difference between the experimental group (cells loaded with genetic circuits) and the control group (unloaded cells) is calculated. The respective metrics are computed as follows:

Option① Growth Rate

Tschirhart et al. investigated the growth rate of V. natriegens in various media and found that the doubling time of the control group under sucrose culture conditions was 28.2±0.1 (min)^[6]. After incorporating the designed genetic circuits, the growth of V. natriegens was simulated using the Michaelis-Menten Kinetic Model, with the growth pattern illustrated in Figure 2. Assuming the bacterial count to be doubled from its initial state, the equation was solved in reverse to obtain an estimated generation time of 53.8 minutes for the experimental group. Substituting this value into Equation (1.2), Tg was calculated to be 90.78%.

Option② Survival Rate

Colony-Forming Units (CFU) are commonly used to estimate the number of viable bacteria or fungi in a sample, serving as a measure of the proportion of cells that maintain normal physiological functions under specific conditions. Conley et al. determined that the bacterial survival count for the wild-type V. natriegens carrying an empty vector after 5 days was 9.8×10⁵±1.1×10⁵ CFU/ml, while the experimental group carrying the mtrCAB plasmid had a 5-day bacterial survival count of 6.3×10⁴±1.3×10⁴ CFU/ml^[7]. Substituting these values into Equation (1.3), Ts was calculated to be 93.57%.

Combining the above analyses, the weighted toxicity value (T_total) was calculated by multiplying the indicator values by their corresponding weights:

Compared to the chassis cells without loaded genetic circuits, the insertion of genetic elements inevitably introduced some toxicity (T_total greater than 0). However, T_total value was less than 1, indicating that the relative toxicity was within onefold of the control group, reflecting that the toxic effects were controlled within a relatively safe range. Upon reviewing the design of the genetic circuits, several aspects can be identified that contributed to reducing the toxicity to the chassis cells. Firstly, the entire system was designed as multiple relatively independent modules, each focusing on executing specific functions. This modular design minimized potential conflicts between different modules. Secondly, the genetic circuits incorporated various inducible promoters that initiate corresponding gene expression only under specific environmental conditions, helping to maintain a low metabolic state in non-target environments and thus avoiding the accumulation of cellular toxicity.

We have found that previous iGEM teams have given little consideration to the issue, however it is an unavoidable problem in synthetic biology. Our estimation of the toxicity of genetic lines is still at a relatively preliminary stage, and we look forward to working with other iGEM teams to find more reliable calculations in the future.

Safety in the oceans

With more than 70% of the Earth's surface consisting of oceans, the effect of the globalization of LuminAid products in the oceans is particularly important. However, due to the special characteristics of the marine environment, it is difficult to intervene effectively in the event of an accident, so the safety of LuminAid products in the ocean requires multiple considerations. We have taken the three main phases of our product line as a whole and have worked to improve the overall safety of our products in every detail of each phase. We manage the potential risks of our products in the following ways: (1) ensuring that the products work properly in the marine environment;(2) preventing the leakage of engineered bacteria into the natural environment and causing contamination;(3) maximizing the protection of the users of the products.

1.Safety in product formation

(1)Bacterial strain selection

Influenced by the glowing sea phenomenon, we are exploring the use of luminescent bacteria. However, for ocean rescue purposes, our engineered bacteria must not only be capable of glowing, but also need to have the following characteristics:

①Rapid growth and the ability to multiply extensively to create a large glowing area in a relatively short time.

②Tolerance to the high osmotic pressure of the ocean environment and the ability to function normally in seawater.

③Non-pathogenic properties that do not pose a threat to individuals in the water.

We compared several marine luminescent bacteria with commonly used chassis bacteria:

	Vibrio fischeri	Vibrio qinghaiensis	Vibrio harveyi	E.coli	Vibrio natriegens
Advantages	Self-luminous; Ocean tolerant; Non-pathogenic	Self-luminous; Non-pathogenic	Self-luminous; Ocean tolerant;	Widely used chassis bacteria	Rapid growth; Ocean tolerant; Non-pathogenic
Shortages	Luminescence relies on quorum sensing	Not adapted to the sea	pathogenic	Not adapted to the sea; non-self-illuminating	non-self-illuminating

All luminescent bacteria use a similar system that oxidizes FMNH2 and long-chain aldehydes with oxygen and luciferase, emitting blue-green light at 490 nm. The luciferase enzyme has alpha (42 kDa) and beta (37 kDa) subunits, both needed for activity, and works best at 18°C but deactivates above 25°C. The lux genes (luxC, luxD, luxA, luxB, luxE) are expressed during bacterial growth, with LuxA and LuxB coding for the luciferase subunits, and LuxC, LuxD, LuxE coding for fatty acid reductase, acyltransferase, and ATP synthase, respectively. These form a complex that produces aldehydes for luminescence.

We have cultured Vibrio fischeri and found it has a slow growth rate and Quorum sensing-dependent luminescence, which is unsuitable for rapid search and rescue needs. Instead, we decided to chose Vibrio natriegens, the fastest-growing bacterium, as the chassis and planned to transfer genes for luciferase (luxA, luxB) and long-chain aldehyde reductase (luxC, luxD, luxE) into it.

In the ocean, the lack of a carbon source can largely limit the growth and luminescence of engineered bacteria. Through literature research, we have identified an engineered modified cyanobacterium that can photosynthesize and release sucrose, and has been shown to coexist better with and provide a carbon source for Vibrio natriegens.

However, the commonly used sump cyanobacterium PCC 7942 is not suitable for the high salinity environment of the ocean, and we chose another sump cyanobacterium PCC 7002 that is tolerant to high salinity.

(2)Preservation of the product: lyophilized powder

Our life jackets need to be stored for long periods of time in scenarios such as boats and airplanes, which poses a challenge to preserve engineered bacteria for long periods of time and keep them active, and we compared several existing methods of bacterial preservation:

	Cryopreservation	Lyophilized preservation	Agar slant culture	Oil Seal Preservation
Advantages	Can be stored for several years; Suitable for most bacteria	Can be stored for several years; Stable and easy to transport; No need for a continuous energy supply	Simple method; Low cost	Can be stored at room temperature; No special equipment required
Shortages	Require special equipment; costly	The lyophilization process may result in the death of a portion of the bacteria	Requires culture medium	Short retention time; Requires culture medium

Eventually, in order to achieve long-term storage and portability, we chose the lyophilized preservation method and prepared lyophilized bacterial powder and tested the activity of lyophilized bacterial powder after resuscitation in the laboratory.

(3)Fluorescent Positioning life jackrt

In order to achieve the goal of making life jackets, we selected a variety of materials. You will find more deteils on our pruduct page.

(4)Packaging of the product

Polypropylene-based nanofibers and electrostatic spinning technology are used to construct packaging for our products. More details could be found on our pruduct page.

2.Safety in Product Applications

(1)Engineered bacteria survive:

The main component of LuminAid is an algal-bacterial system. It is crucial to ensure the safety of the product by maintaining the survival of the microorganisms and preventing any negative effects caused by their death. We have selected Vibrio natriegens and cyanobacterium, both of which are marine microorganisms, with the ability to survive in high osmotic environments and low temperatures. However, the low temperature of seawater may increase the content of ROS in Vibrio, which can be detrimental to its survival and function. Therefore, we need to enhance the cold tolerance of Vibrio natriegens to ensure its effectiveness.

Based on the coexistence of algae and bacteria, we utilized the sucrose produced by algae in an innovative way. Firstly, the sucrose serves as a carbon source, enabling the long-term survival of Vibrio natriegens. Secondly, we designed a gene circuit where the continuous supply of sucrose activates PsacB, leading Vibrio natriegens to express superoxide dismutase (SOD) and catalase, which break down ROS into water and oxygen, thereby preventing oxidative stress and conferring cold tolerance to Vibrio. The cold-tolerant Vibrio can also be used in spill containment.

Cyanobacterium can determine the source of sucrose for the sodium-demanding Vibrio, and its survival is equally important. As a member of the cyanobacterial family, Polychlorophylla is well adapted to the marine environment, with a high cold tolerance and a salt tolerance that varies from strain to strain. For our products, we have selected the salt-tolerant strain PCC7002 to enhance its salt tolerance. In this way, the survival of microorganisms in our algae-bacteria coexistence system is fully guaranteed.

(2)Prevent leakage of engineered bacteria:

There is the potential for leakage of our engineered bacteria in the ocean, and if leaked into the marine environment, we believe there would be two risks:

Risk of false positives: uncontrolled growth of the engineered bacteria after leakage creates a fluorescent area that is too large and no longer helpful for accurate location determination.

Risk of bacterial contamination: Although Vibrio natriegens is of marine origin and is not pathogenic to humans, the leakage of engineered Vibrio natriegens has implications for the Earth's ecology and may cause some unknown ecological damage.

In the ocean, traditional sterilization methods are no longer suitable because of the tedious steps required. Kill-switch circuit that have been widely used by previous researchers and iGEMers are considered preferred.The main body of the kill-switch circuit usually comprises two essential components: a sensor and a suicide effector. The sensor detects environmental changes that can activate the suicide effector thus inducing apoptosis.

We found that the methodology of the microbial survival module can also be used as part of the leakage prevention module. When Vibrio natriegens accidentally leaks out of the algal-bacterial coexistence system, the sucrose concentration around the bacteria decreases, and there is a lack of carbon supply to support survival. At the same time, the cold-tolerant module will also fail. Although Vibrio natriegens grows fast and survives well, the lack of cold-tolerance will also lead to the death of the engineered bacteria due to the accumulation of ROS.

We thought further about the leakage prevention module and concluded that the leakage prevention method relying on the microbial survival module is not active, after all, there may be other carbon sources supplied in the ocean, and the effect of the killing method of temperature-dependent ROS accumulation is not necessarily significant. We therefore designed active killing lines that can sense changes in sucrose concentration to secrete toxic proteins.

We designed two circuits for analysis. Firstly, for circuit 1 we used PSacB to sense sucrose concentration and set the deterrent protein lacI to control the expression of MazF toxin protein afterward. Circuit 1 decreases the secretion of lacI when the sucrose concentration decreases around the engineered bacteria, thus weakening the deterrence of Plac by lacI, which in turn expresses MazF to kill the bacteria. However, there is a problem that it may lead to false killing before our product is put into use, and the expression of PSacB was not found to be high in the Nanjing-China 2022 IGEM team. Of course line one has already realized the judgment of whether the engineered bacteria are out of the coexistence system based on the presence or absence of sucrose, and we then iterated on this basis.

Circuit 2 was improved according to line 1's sucrose sensing, so that the engineered bacteria activated their sensing of sucrose only in the hypertonic environment of the ocean, and after entering the ocean, the spare sucrose in the product and the sucrose produced by the algae inhibited the deterrence of Pscr by ScrR, resulting in the expression of lacI and the non-expression of MazF. After the engineered bacteria were detached from the system, the sucrose concentration decreased, the deterrent effect of ScrR on Pscr was enhanced, the expression of lacI decreased, and MazF was then expressed to play a killing role.

In this way, our anti-leakage module was perfected. When leakage occurs, it can not only passively kill through the reverse side of the microbial survival module, but also actively kill by sensing the change of sucrose concentration, forming a comprehensive passive-active anti-leakage module.

3.Safety in product recycling and disposal

The success of Luminaid in assisting rescuers in locating and rescuing people in distress does not mean the ultimate success of the product. Proper disposal of the engineered bacteria to prevent contamination of the environment is an issue that must be considered. In this regard, we have designed two recycling options for Luminaid:

Option①:Kill the engineered bacteria that have fulfilled the task. We design a temperature control system and a drug control system to promote the engineered bacteria toxin protein to trigger bacterial death by controlling the temperature or adding inducers.

Drug control system: We initially chose two commonly used inducers: arabinose and rhamnose, however, when comparing the properties of these two inducers we found that: rhamnose degrades slowly in the natural environment and may be present in the water column for long periods of time, causing potential bioaccumulation and environmental pollution. Rhamnose may have negative effects on aquatic organisms, especially microorganisms and aquatic plants. High concentrations of rhamnose may interfere with the physiology and ecosystem functions of aquatic organisms. Compared to rhamnose, arabinose is generally considered to have less or negligible impacts in the marine environment. Arabinose is a naturally occurring simple carbohydrate that can be utilized as a carbon source by many microorganisms and aquatic organisms. It is usually well biodegradable and can be rapidly broken down and utilized by microorganisms. It is a natural product that does not usually cause discomfort or toxic reactions in living organisms, is usually released in small amounts in the marine environment, and can be rapidly broken down by microorganisms in the water without causing long-term accumulation or contamination problems. Therefore, we believe that arabinose is a better choice. However, further experiments are still needed to prove whether the inducer can effectively kill engineering bacteria in seawater.

Temperature control system: suicide is triggered by heating the engineered bacteria to 37°C, inducing the expression of the toxin protein mazF. Limited by the specific heat capacity of seawater, the heating time may be longer, so the specific performance of this system needs to be further explored.

Option②: Recycle the engineered bacteria that have completed the task and re-make them into lyophilized bacterial powder. This is a more economical recycling method, but repeated freeze-drying will cause the engineering bacteria to lose their activity. In the future, we will test the effect of multiple freeze-drying-storage-recovery on the activity of the engineering bacteria, and calculate a reasonable number of times of recycling in order to ensure the quality of the product.

Safety in space

Our knowledge of the space environment is limited compared to our understanding of the oceans, making space exploration a major challenge. The unique environments of different planets in space pose significant challenges to our safety efforts.

Mars, as the closest red neighbor to Earth in the solar system, is increasingly seen as a potential location for human settlement due to its unique natural environment and potential resources. However, Mars presents difficulties such as a thin atmosphere, large temperature differences between day and night, and high levels of cosmic ray radiation. Therefore, the development of Mars infrastructure is essential for future landing and migration. As a result, we have selected Mars as the first target for our project.

According to NASA experts, the Mars base must follow the principle of “space”, wear spacesuits when going out, and keep communication with Earth open at all times. The project involves engineering bacteria with the ability to withstand strong ultraviolet radiation, low temperatures, and dryness on Mars. Additionally, solutions for preventing leakage and infection of engineering bacteria due to the microgravity environment in space are being considered.

We compared the strategies thought to be proposed by the iGEM team to inform the safety module of this and future projects in the space village.

1.Strong radiation from space threatens engineered bacteria

Mars has a weak global magnetic field (≤5 nT)^[14](Figure 3). There is no shielding against solar cosmic rays (SCRs), galactic cosmic rays (GCRs), etc., so the radiation environment on Mars's surface is harsher than Earth's. With solar activity and the weakening of the heliospheric magnetic field, the GCR radiation on the surface of Mars can increase by about 50%(Figure 4).

Exposure to radiation can lead to bacterial mutation or death. Ultraviolet radiation can cause DNA breakage, leading to the death of engineered bacteria and loss of their luminescent function. It can also cause mutation of functional genes, potentially resulting in the acquisition of genes related to virulence and drug resistance. Microgravity can weaken astronauts' immune systems, making it easier for bacteria to adhere to the human body and endanger the astronauts' health.

To counteract the damaging effects of UV radiation, it is important to protect the engineered bacteria to prevent infection from mutated pathogenic bacteria. The bacteria we have selected, Chlamydomonas reinhardtii, can form large colonies and survive in space as aggregates, exhibiting resistance to environmental hazards such as UV radiation. Literature suggests that these colonies can survive on the ISS for 15 to 45 years.

In order to further improve the radiation resistance of the engineered bacteria, we envisioned to introduce RecO and RecF in radiation-resistant Chitinobacterium, and successfully verified in E.coli that the survival rate of the bacteria under UV irradiation increased after the introduction of RecO and RecF plasmids. From the physical isolation point of view, we designed special space suits containing radiation protection layer, which can simultaneously reduce the exposure risk of astronauts and engineered bacteria during extravehicular activities or encountering sudden radiation events.

In addition, we reviewed other teams' methods for safeguarding the survival of engineered bacteria through radiation resistance.

(1)Melanin

The team iGEM2009_Tokyo_Tech introduced melanin into E. coli for the first time by overexpressing tyrosinase, which catalyzes the conversion of tyrosine to dopa kun, followed by a non-enzymatic chain reaction that allows melanin to accumulate. By blackening E. coli, they hope to increase heat and UV absorption, thus reserving heat for bacterial growth and reducing the risk of UV damage.

(2)Ultraviolet light damages nucleic acid endonucleases

The team iGEM2014_Stanford-Brown-Spelman isolated the radiation resistance gene uvsE from D. radiodurans genomic DNA, which encodes the UV DNA damage endonuclease uvsE that catalyzes the excision and repair of pyridine dimers after UV irradiation. They transformed it into DH5-α cells and exposed them to 254 nm UV light. Their experimental results showed that uvsE significantly enhanced the radiation resistance of DH5-α cells and determined that the D. radiodurans UV-damaged nucleic acid endonuclease uvsE gene confers additional radiation resistance to E. coli.

(3)DNA repair proteins FEN1 and XRCC1

The FEN1 protein, from the XPG/RAD2 family of nucleic acid endonucleases, is one of the 10 essential proteins for free DNA replication, and is involved in resolving 5' protruding 'flaps' formed when DNA strands fail to bind correctly, in managing the 5' end of Okazaki fragments in lagging-strand DNA synthesis and in seamless substrate transfer to enzymes in long patch base excision repair. XRCC1 is primarily involved in DNA repair of radiation- and alkylating agent-induced single-strand breaks, and usually performs base excision repair in conjunction with DNA ligase III, polymerase β, and poly(ADP-ribose) polymerase.

The team iGEM2023_Fudan expressed codon-optimized FEN1 and XRCC1 in E. coli hospitals, and experimental results showed that the two together conferred stronger UV resistance to E. coli.

(4)Mycosin-like amino acids (MAA)

Team iGEM2022_LINKS_China has introduced mycosporine-like amino acids (MAA) into Saccharomyces cerevisiae to absorb the sun's ultraviolet rays, producing a bioproduct alternative to sunscreen. MAA can be produced by algae such as cyanobacteria and red algae, as well as other marine organisms exposed to strong UV light, but the source organisms are slow-growing, whereas S7P catalyzes the production of MAA through a series of enzymatic reactions, and S7P itself is made via the pentose phosphate pathway from glucose. So the team inserted xylose (Xyl1, Xyl2, Xyl3) from Scheffersomyces stipitis into Saccharomyces cerevisiae to enable it to take up xylose and produce X-5-P, which can be directly converted to S7P, and knocked out the downstream gene, TAL 1, to prevent S7P from being converted to anything else, and inserted Nostoc punctiforme, Nostoc linckia, and Nostoc linckia. Nostoc punctiforme, Nostoc linckia and Actinosynnema mirum to produce MAA rapidly. However, the team mentioned that MAA cannot completely absorb all UV radiation from the sun, and some of it will still escape, which cannot be ruled out as harmful to the engineered bacteria.

2. Extreme cold temperatures in space hinder survival of engineered bacteria

Mars has a thin atmosphere and is farther from the Sun, so its surface temperature is much lower than Earth's. The average surface temperature on Mars is -46°C, and can drop to as low as -143°C at the poles in winter, and rise to about 35°C at the equator at noon in summer. Only cold-loving bacteria can survive at these extremely low temperatures. We discovered that Deinococcus radiodurans, which is resistant to radiation, stop growing when the temperature is lower than 4°C or higher than 45°C. Therefore, it's crucial to modify engineered bacteria to withstand the very low temperatures of Mars.

We summarize the contributions made by previous iGEM teams to this area.

(1) Late embryogenesis-enriched (LEA) proteins

Team iGEM2010_Valencia/lea expressed an osmotic stress-regulated "antifreeze protein", the late embryogenesis-enriched LEA protein, which enables E. coli to resist cold shock. Changes in chemical potential and osmotic pressure when the bacteria are exposed to low or high temperatures result in the removal of liquid water from the cell, which activates LEA, causing the LEA protein to accumulate in nutrient tissues during osmotic stress, which in turn is involved in retaining water, preventing the aggregation of cellular proteins and the crystallization of other molecules during cellular desiccation, and stabilizing the cell membrane. The team selected the soybean PM2 (LEA3) protein and introduced it into E. coli to simulate cycling temperatures during a Martian solar day to compare E.coli survival. The results of the experiments showed that LEA was protective and synergistic with glycerol.

(2)Antifreeze Protein (AnAFP)

Team iGEM2023_Fudan heterologously expressed an antifreeze protein (AnAFP) derived from Ammopiptanthus nanus in Escherichia coli to promote its survival in sub-zero environments. AnAFP prevents macromolecules from solidifying by binding to or dissociating from calcium ions, and protects the stability of membrane structures under cold conditions by binding to or dissociating from membranes. The experimental results show that after 48 hours below freezing, the AnAFP-transformed E.coli showed significant freezing tolerance compared with normal E.coli.

3.Future risks that still need to be addressed

Dusty Martian atmosphere affects visibility of engineering bacteria

Mars atmosphere is composed of about 95% of carbon dioxide, 3% of nitrogen, 1.6% of argon, very little oxygen, water vapor, etc., but also full of a lot of 1.5 microns in diameter or so of suspended dust, absorbing blue light to make the sky into a yellowish-brown^[14]. As a desert planet, Mars surface dunes, gravel everywhere, there is no stable liquid body of water, sand and dust suspended therein, dust storms often occur every year. This greatly reduces the visibility of the engineered bacteria that emit fluorescence to be recognized.

Engineered bacteria corrode space suits

We apply engineered bacteria to the surface of spacesuits to become a special luminescent material. However, once the engineering bacteria are leaked, it is easy to form biofilm in the spacesuit, and the microbial growth, reproduction and metabolism will corrode the spacesuit and even infect the astronauts. Therefore, to prevent the leakage of engineering bacteria, the use of antimicrobial materials for spacesuits, such as nano-antimicrobial insulating polymer composites and long-lasting antimicrobial coatings on metal surfaces, can be considered; in addition, bacteria can also be packaged with a hydrogel layer to prevent the leakage of engineering bacteria and to control microbial sources.

Safety in HP

1.Education

During the advancement of our project, we have consistently maintained attention to the social hotspots related to us.

Faced with the frequently occurring maritime accidents, which bring about substantial economic losses and heavy casualties, they constitute a severe reality that cannot be overlooked. These tragedies ruthlessly reveal a fact: maritime disasters are not distant concepts but can threaten the safety of us and those around us at any moment. Hence, in addition to conducting detailed statistical and in-depth cause analysis to draw lessons and take preventive measures, pre-event publicity, education, and popular science promotion also hold a pivotal position. We are deeply aware that in emergency situations, the correct handling methods and self-rescue knowledge can significantly enhance an individual's probability of survival. Therefore, when designing our products, apart from clearly introducing the product structure, usage methods, and product features in the product manual, we particularly intend to add a paper version of the "Maritime Self-Rescue Guide" in each product. Maybe in some emergency circumstances, we expect it to play a significant role.

In addition, our team has been actively involved in scientific communication and educational promotion to raise public awareness. By sharing knowledge, we are committed to raising awareness and understanding of the correct self-rescue methods during drowning.

We have written a scientific article titled "Stay Calm at Sea: Secrets of Self-Rescue Help You 'Sail Through the Waves'" and published it on our official account.

Meanwhile, we attach great importance to "self-education". After the incidents of "Girl Surviving 36 Hours Drifting at Sea", our team organized collective learning of this news and shared our own thoughts.

"Recently, a Chinese national was swept away by the sea while enjoying leisure time on a beach in Japan. The embassy promptly coordinated with local law enforcement to initiate a comprehensive search and rescue operation. Japanese authorities, along with various departments, collaborated in the joint search effort, and a compassionate cargo ship came to assist. Ultimately, the citizen was successfully rescued."

This news story has prompted us to heighten our awareness of safety and underscored the critical importance of life support systems in space, as well as the necessity for enhancing search and rescue capabilities at sea. The news also reaffirmed the mission of our project.

2.Communication

Specialization: During the Synbio Challenges, the judges asked questions about our project, and our solutions were presented as follows:

In our discussions with the BNDS-China team, we all agreed that more extensive safety measures were needed to manage any risks associated with the project.

At the same time, we learned from and discussed with the BNDS-China team how to apply molecular docking technology to verify the affinity between the regulatory protein ScrR and the target promoter, in order to further confirm the feasibility of our passive-active anti-leakage circuit.

We were curious whether CHINA-FUZHOU had considered the toxic effects of loading and expressing genetic circuits on the chassis cells. After considering, CHINA-FUZHOU indicated that one of the products they expressed, carotenoids, could enter the plant's own metabolic pathway, which provided us with a new perspective.

On September 17th, we participated in "Navigating the future of AI in synthetic biology", where speakers discussed the safety issues of AI in its application in synthetic biology. As the use of AI continues to rise, its safety is becoming increasingly important.

To ensure the healthy development and widespread application of AI technology, the following suggestions are put forward:

Firstly, enhance the application of AI in risk assessment and prediction. Utilize AI technology to predict and evaluate potential risks in synthetic biology, such as off-target effects and ecological impacts in gene editing and new organism design, etc. Develop a risk warning system based on big data and machine learning, and monitor and analyze data in real time to issue early warnings for potential safety issues. Specific measures include building a risk assessment and prediction system, covering data collection (establishing a synthetic biology database), model development (using machine learning and other technologies), and warning mechanisms (promptly issuing warning signals and responding to them).

Secondly, enhance the role of AI in biological safety regulation. Utilize AI to assist regulatory agencies, improving the efficiency and accuracy of regulation, and building an intelligent biological safety management system to monitor biological samples and experimental processes comprehensively. Practical measures include building an intelligent regulatory system, including the development of regulatory platforms (to achieve remote monitoring and data analysis) and automated detection (using image recognition, etc.) as well as feedback mechanisms (to promptly address issues and ensure the effectiveness of regulation).

Third, promote the ethical integration of AI and synthetic biology. Pay attention to ethical issues in the development and application of AI, strengthen ethical education for researchers and the public, and promote healthy and sustainable development. Measures include ethics education and standard formulation, such as integrating ethics education into curriculum systems to enhance researchers' awareness, organizing experts to formulate standard norms, and promoting publicity and popularization through media and networks.

Fourth, promote international cooperation and standard setting. Strengthen international cooperation and exchange to address global challenges and promote the formulation of international standards. Measures include actively participating in exchanges to share achievements, jointly conducting research projects, and promoting the formulation of unified standards.

Policy analysis

We have organized the policies pertaining to the project into the table below:

Perspective		Specific terms	Countermeasure
Product: Life Jacket	Life jacket construction: buoyancy section	Article 22 of the International Convention for the Safety of Life at Sea: Recognized lifejackets should be able to support the face of a person who is exhausted or unconscious in the water and keep their face above the water by tilting their body vertically backward. Clause 5.3.4 of GB/T 32234.1—2024, "General Requirements for Personal Flotation Devices - Part 1: Lifejackets for Ocean-going Vessels": The total buoyancy of adult (user weight > 43 kg) lifejackets tested according to ISO 12402-9 shall not be less than 150 N.	The buoyancy equipment with conventional life jackets needs to be genetically modified by Vibrio Natriegens to express gas vesicle proteins, which will enable the bacteria to float more on the surface of the sea, expanding the visible area of water glow while also optimizing the buoyancy performance of the life jacket to some extent.
	Life jacket construction: illuminating section	5.4 "Visibility" requirements in "Personal Flotation Devices - Part 1: Lifejackets for Ocean-going Vessels - Safety Requirements" (GB/T 32234.1-2024): The area of reflective material on the surface of the lifejacket should not be less than 400cm², so that it can be seen from all directions around and above the lifejacket when the wearer is in a relaxed state in the water.	The algae-fungus lyophilized powder can form visible blue fluorescence when it comes into contact with water, which can help rescue teams locate missing persons better under poor lighting conditions at night on the basis of reflective materials.
	Life jacket material: standard for wound dressing testing	"Test Methods for Contact Wound Dressings - Part 1: Liquid Absorbency" (YY/T 0471.1-2004) includes: liquid absorbency, water vapor permeability of breathable film dressings, water resistance, comfort, antibacterial properties, and odor control.	Taking into account that open wounds exposed to seawater for a long time can cause sea immersion injuries, we thought about combining the inner layer of the life jacket with wound dressings, using materials that adhere well to the skin, have excellent antibacterial and thermal properties.
	Life jacket materials: standard for the development of nanomaterials	The "Part 1: Framework for the Evaluation of Safety and Effectiveness of Medical Devices Containing Nanomaterials in Clinical Application" of the "Guidelines for the Evaluation of Safety and Effectiveness of Medical Devices Containing Nanomaterials" states: "Section V, Biological Evaluation: When nanomaterials are used as coatings for medical devices, the risk of shedding, degradation, and release should be considered. When conducting biological evaluations, the following factors should be taken into account: cytotoxicity, irritation, sensitization, systemic toxicity, pyrogenicity, implantation, hemocompatibility, genotoxicity, carcinogenicity, and reproductive toxicity."	Our product has an outer layer made of nanofiltration membrane produced by electrospinning technology, and we have taken into account the risk of bacterial leakage due to material wear. An anti-leakage module was incorporated into the design of the circuit during the process. ·If it is put into use in the future, after the product positioning and carrier form are clarified, a series of tests and evaluations will be needed.
Microbiological Safety	Classification of Microbes	The seventh article of the Regulations on the Biosafety Management of Pathogen Microorganisms Laboratory (2018 Revised) states: The State shall classify pathogenic microorganisms into four categories based on their infectivity, the degree of harm they cause to individuals or groups after infection, and whether they are microorganisms that have not been found or have been declared eradicated in China: Class I pathogenic microorganisms refer to microorganisms that can cause very serious diseases in humans or animals, as well as microorganisms that have not been found or have been declared eradicated in China. Class II pathogenic microorganisms refer to microorganisms that can cause serious diseases in humans or animals and are easily transmitted directly or indirectly between people, animals, and environments. Class III pathogenic microorganisms refer to microorganisms that can cause diseases in humans or animals, but usually do not pose serious harm to humans, animals, or the environment, have limited transmission risks, rarely cause serious diseases in laboratory infections, and have effective treatment and prevention measures. Class IV pathogenic microorganisms refer to microorganisms that usually do not cause diseases in humans or animals. Class I and Class II pathogenic microorganisms are collectively referred to as highly pathogenic microorganisms.	The microorganisms used in our project belong to Class 4 pathogens: they refer to microorganisms that are unlikely to cause disease in humans or animals under normal circumstances.
	Development Process of Microbiological Products	Article 35 of the People's Republic of China's Biological Safety Law stipulates that units engaged in biotechnology research, development, and application shall be responsible for the safety of their own biotechnology research, development, and application, and shall take biological safety risk control measures, establish work systems for biological safety training, follow-up inspections, and periodic reporting, and strengthen process management. Article 42: The State shall strengthen the management of biological safety in laboratories for pathogenic microorganisms, and formulate unified standards for laboratory biological safety. Pathogenic microorganism laboratories shall meet the national standards and requirements for biological safety. Experimental activities involving pathogenic microorganisms shall be strictly observed in accordance with relevant national standards and laboratory technical specifications, operating procedures, and safety prevention measures.	We have uniform laboratory biosafety standards and have also implemented appropriate safety measures to control waste that may contaminate the environment.
	Genetic Engineering	Article 6 of the Regulations on Safety Management of Genetic Engineering: According to the potential degree of danger, genetic engineering work is divided into four safety levels: Safety Level I, where genetic engineering work poses no danger to human health and ecological environment; Safety Level II, where genetic engineering work poses low degree of danger to human health and ecological environment; Safety Level III, where genetic engineering work poses moderate degree of danger to human health and ecological environment; and Safety Level IV, where genetic engineering work poses high degree of danger to human health and ecological environment.	The microorganisms used in our project are generally non-pathogenic to humans and pose no threat to human health and the ecological environment. They are classified as safe level I.
Environmental Ecology	Oceans: Microbial leakage and Marine pollution	Article 22 of the Law of the People's Republic of China on Marine Environmental Protection: Enterprises, institutions and other business operators shall give priority to using clean and low-carbon energy sources, adopt clean production processes with high resource utilization rates and low emissions of pollutants, and prevent pollution of the marine environment.	Our products feature a dual-layer filter membrane structure that prevents algae and bacteria from passing through, making it less likely for microbial contamination to occur. The bottom layer of the filter is made up of Synechococcus and Na nitrosomonas, which are naturally occurring marine organisms that produce the non-polluting substance sucrose, resulting in less environmental pollution.

Dual-use analysis

The characteristic of science and technology, which can pose a security threat while promoting the development of human society, has been referred to by academics as "dual-use". The concept of "dual-use technology" initially referred specifically to technologies that had both actual or potential military and civilian applications.

The U.S. Congress in the Defense Industry Technology Conversion, Reinvestment and Transition Act for the first time on the "dual-use technology" clearly defined: “dual-use technology” is related to products, services, standards, processing or procurement, can meet the military applications and non-military applications, respectively. A report by iGEM Team Bielefeld-CeBiTec 2015 also addresses the 2 sides of biotechnology. With this in mind, we assessed this project with a careful dual-use analysis.

1.Possible benefits of the project

(1)Good biological adaptability

The symbiotic system of Vibrio natriegens and Synechococcus has good biological adaptability. The ability of the symbiotic system to adapt to hypertonic cold seawater is improved by the cold-tolerant safety module, and at the same time, it can express antimicrobial substances to reduce the effect of bacteria in the ocean on wounds.

Using synthetic biology methods to introduce recO and recF into symbiotic system, an efficient DNA repair system is established in the cell, which can rapidly repair DNA molecules damaged by UV radiation and protect the integrity of the genome.

(2)In-situ resource utilization

①The symbiotic system can better adapt to extreme environments and cope with harsh environments such as coal mines, polar regions, and the deep sea.

②Provide the ability to utilize resources: use solar energy to produce sucrose, glow in emergency situations and provide help for rescue.

(3)Life Support

①LuminAid aims to utilize synthetic biology to design a lifejacket with life-support functions to achieve resistance to low temperature, high temperature, acid and alkali, and high pressure to ensure that it can function in extreme environments.

②Algae-bacteria symbiotic systems are in extreme environments like space, processing, regulating and recycling air, water and solid waste to create a mini-ecosystem.

③Provide nutrients and assist in nutrient recyclin.

2.Potential risks of the project

(1)Potential risks of chassis organisms

The chassis organisms Vibrio natriegens and Synechococcus sp. PCC7002 have good stability and rarely have an abnormal increase in toxin genes. In addition to this, the inserts during the experiment do not confer characteristics that could cause harm to the chassis organisms. Based on the characteristics of the final product, it is expected that the project involves low potential risk.

All of our experiments are strictly limited to the laboratory and no organisms will be released in the natural environment.

(2)Potential risks of information and technology

There is a low risk of potential misuse of knowledge, information and technology regarding this project. All methods used in the project are basic molecular biology techniques such as plasmid extraction, RNA extraction, agarose gel electrophoresis. Digital tools widely used for genetic circuit construction were applied and specifications were followed during the design of the circuits to ensure that there would be no harmful consequences of enhanced toxicity. The project is not developing any new technology that could be of concern for large scale illegal use.

(3)Potential risks of possible misuse

Every coin has two sides. Our engineered bacteria could be used maliciously for genetic coding by people trying to disrupt recombinant DNA technology. In this regard, we will strictly limit the genetic mapping of our engineered bacteria to prevent tampering by unscrupulous people. At the same time, we keep a detailed record of who uses the engineered bacteria, when, and for what purpose, to ensure that controls are in place.

3.Measures to ensure safety

(1)Meticulous design

Radiation sources such as cosmic rays and solar wind exist in space, and for good radiation protection, we introduced recO and recF genes into the engineered bacteria for UV radiation damage resistance. UV-resistant engineering bacteria may have the ability to survive after leakage in space compared to chassis bacteria that have not been introduced with radiation-resistant genes. For this reason, we introduced a comprehensive passive-active anti-leakage module, which is capable of not only passive killing by the reverse side of the microbial survival module, but also active killing by sensing changes in sucrose concentration.

A sucrose dilution suicide switch was designed. Upon the leakage of the engineered bacteria from the biofilm, the sucrose concentration is reduced. Subsequently, the engineered bacteria that had leaked outside the biofilm expressed MazF, which resulted in the bacteria committing suicide. Additionally, the absence of a carbon source is a contributing factor in bacterial death. When combined with the active expression of the toxin protein MazF, this constitutes a reliable strategy to prevent bacterial leakage. Furthermore, we selected drug-controlled and temperature-controlled termination switches to guarantee the active eradication of the engineered bacteria, thus preventing potential leakage and associated complications. In addition to this, it is of equal importance to prevent the release of engineered bacteria into the environment in the context of space exploration. Our protection strategies are equally applicable in this domain and we believe it to be an effective measure in avoiding potential biosecurity issues.

(2)Standardized use and effective supervision

In addition to the basic biosafety measures before entering the lab, the lab personnel also record the procedure of each operation to ensure that all experimental operations are standardized and follow the rules of the BSL2 lab. In addition, our PI has been monitoring our progress to ensure that none of the sequences used in this project are dangerous or pose any risk of misuse.

4.Risks and Benefits Comparison

The benefits of this project outweigh the risks associated with the development of the project. In addition, given the strict compliance of the LuminAid project, the potential for misuse of the technology is low. In summary, we can conclude that this project makes a useful attempt to rescue in extreme environments, especially in space and contributes to the technological development of human society with no serious security threats.