Safety

Introduction

Prioritizing safety is our top concern. Our team has diligently familiarized itself with the nuances of safety associated with synthetic biology, especially focusing on the guidelines and best practices that underscore both biosafety and biosecurity in a laboratory setting.

In the realm of synthetic biology, it is imperative to safeguard not only the researchers within the laboratory but also the broader community and the environment. Additionally, it is crucial for us to address both the potential dual-use implications and the overarching biosecurity concerns surrounding not only our specific project but synthetic biology at large.

Our finalized product consisting of the modified fungus would be employed in agricultural areas, which poses several safety hazards we need to consider and precautions we need to apply.

1. Ecological Impact

The potential introduction of Fungilyzer into the environment poses certain risks. Specifically, there is a concern that it might disturb local ecosystems, which could negatively impact native species or unintentionally provide advantages to invasive ones. To mitigate these concerns, we are committed to conducting detailed preliminary studies in a controlled environment. These studies would take place in contained field trials, ensuring that any potential effects of Fungilyzer are strictly limited to the testing area, minimizing the risk of unintended spread to broader ecosystems. Several key parameters would be assessed during these trials:

Growth rate of other plant species: We would closely monitor the growth patterns of other non-crop plants within the testing area to determine if Fungilyzer affects their development, either positively or negatively.

Soil microbial composition: The balance of microbes in the soil is vital for plant health and overall ecosystem function. We would analyze the soil's microbial makeup before and after introducing Fungilyzer to detect any shifts in microbial populations.

Health and behavior of local fauna: Observing the health, behavior, and population dynamics of local insects, birds, and other animals will provide insights into whether Fungilyzer has any secondary effects on the broader ecosystem.

Spread and persistence of Fungilyzer: It is crucial to understand how long Fungilyzer remains active in the environment and if it spreads beyond the intended application area. This will help determine the frequency and volume of applications needed and any potential risks of contamination to nearby ecosystems.

Following these initial trials, we would ensure regular monitoring post-application in real-world scenarios. This continuous observation would help us detect and address any unforeseen ecological changes, emphasizing our commitment to both innovation and environmental responsibility.

2. Human Exposure

Potential Hazard: There is a concern that workers or individuals in proximity to the fields might inadvertently come into contact with Fungilyzer.

Safety Precaution: To address this, we will be implementing robust safety guidelines for workers, detailing the proper handling and application of the product to ensure minimal risk of exposure. Alongside this, we will be placing clear markings and warning signs around the fields to keep the public informed and cautious. It is worth noting, however, that our product is classified as a Biosafety Level 1 microbe. This means that even if Fungilyzer were to come into direct contact with a human, it is not expected to pose any threat or cause disease in healthy individuals. Nevertheless, our primary goal remains to prioritize safety at all times and reduce any potential risks.

As Fungilyzer is a genetically modified organism coming into direct contact with food crops, it is essential to consider what this will mean to the consumer. The plants themselves would not be genetically modified, and the fungus, based on our system, is likely going to die off during the growing process, but depending on the crop, contamination with GMOs could not be excluded. Since Fungilyzer is applied at the root, it would be safe to consume crops grown above the surface of the soil, especially those growing on the plant itself. Crops that grow on the soil surface, like pumpkins, and those grown in the soil, like carrots, would have to undergo testing. The testing would observe how and if any Funglilyzer survives on the crops and if it could be safely removed. This could be made possible by taking samples, growing them on petri dishes, and performing qPCR on gene-tags. If any contamination should appear, we would have to adopt measures, for example, washing the vegetables and fruits after harvesting, implementing sterilization with UV, or assembling more kill switches. Since our project already includes a gene for controlled cell death, it would be possible to build in further regulations, for example, triggering the death gene at low temperatures so the remaining cells would die off at the latest during the storage of the fruits and the remains could be safely washed off without any contamination. Another option would be to add a kill switch based on other non-harmful substances that could be used to easily and safely treat the harvested crops. If it turns out that our GMO has any consequences for any living beings or the environment, we would stop the use immediately and ensure that any sold product with our fungus does not come to any further use.

3. Water Contamination

Potential Hazard: Fungilyzer could potentially leach into the water table, contaminating local water sources.

Safety Precaution: We designed our product to degrade naturally over time using its inbuilt kill switch, reducing the risk of water contamination. Moreover, we would implement buffer zones near water sources to minimize potential runoff.

4. Airborne Spread

Potential Hazard: Particles of Fungilyzer could become airborne and spread to unintended areas.

Safety Precaution: Application guidelines would be established to minimize aerosolization, such as using specific application methods or applying the product during specific weather conditions.

5. Interaction with Other Agricultural Products

Potential Hazard: Fungilyzer might interact negatively with fertilizers, pesticides, or other field-applied products.

Safety Precaution: Before introducing Fungilyzer to the market, compatibility tests would be conducted with a wide range of commonly used agricultural products to ensure safety and effectiveness. The following test would need to be performed to assess Fungilyzer compatibility with other agricultural products.

• Chemical Interaction Tests: This would assess if Fungilyzer reacts chemically with other products. The goal is to ensure that no harmful by-products are formed when they coexist.

• Physical Compatibility Tests: This would evaluate if Fungilyzer can be mixed directly with other products without causing issues like precipitation or separation. This is important for farmers who might want to apply multiple products simultaneously.

• Microbial Activity Tests: Given that Fungilyzer is a microbe, it is essential to check if other products inhibit or promote its growth and activity. This ensures that Fungilyzer functions optimally in varied environments.

• Residue Analysis: This evaluates the residues left on crops post-application. It is crucial to ensure that residues, especially when Fungilyzer is used alongside other products, are within safe limits and do not pose a threat to consumers.

Farmers and field workers would be equipped with a comprehensive list detailing which products should not be used in tandem with Fungilyzer to prevent any negative interactions.

6. Persistence in the Environment

Potential Hazard: If Fungilyzer remains active in the environment longer than intended, it could have unforeseen impacts.

Safety Precaution: Our product has been designed for controlled degradation. Post-application monitoring would ensure that it breaks down as expected.

7. Wildlife Interaction

Potential Hazard: Local wildlife might consume or come into contact with Fungilyzer, leading to unforeseen ecological effects.

Safety Precaution: Warning signs and barriers would be used to deter wildlife from treated areas, where applicable.

In addition to those, we also have to look at safety and security in and around our laboratory, as well as for the researchers working in it. Our team underwent comprehensive training on June 20 and 23 to ensure we worked safely and efficiently within the laboratory.

Furthermore, in our commitment to safety, we have actively consulted with iGEM on risk management measures, including the submission of check-in forms and direct communication with relevant committees. Additionally, we have sought advice from other specialists, such as institutional biosafety officers, to ensure a comprehensive approach to safety.

Safety Outreach

We extended the conversation beyond the confines of our laboratory, engaging in meaningful discussions on biosafety, biosecurity, and dual-use concerns with fellow researchers and the general public. Especially regarding dual-use of technologies in synthetic biology, we found the following points as being useful approaches:

Monitor research that has both civilian and potential military applications. Restrict the publication of certain research details if they might aid in misuse. Regulate and monitor the sale and distribution of specific tools, materials, and organisms used in synthetic biology. Use traceable markers or tags in synthetic DNA sequences to track their origin and distribution. Work on global standards and best practices for synthetic biology through international bodies and agreements. Share information on potential threats and misuse patterns. Establish ethics committees to review and oversee synthetic biology projects, especially those with potential dual-use implications. Educate policymakers, law enforcement, and the public about the potential and risks associated with synthetic biology. Develop systems to quickly respond to any accidents, breaches, or misuse in the field.

Environmental Safety Measures

Within our laboratory, when handling genetically modified or potentially harmful microorganisms, we employ biohazard containers for safe disposal. We utilize two distinct categories of sharp-safe containers: plastic bags for non-sharp biological refuse and sturdy containers for sharp waste materials. After each use, all tools and reusable laboratory equipment undergo autoclaving. To minimize the risk of cross-contamination, we designated specific zones for "used" materials, ensuring they remain separate from uncontaminated or "clean" zones.

Saccharomyces cerevisiae

For our final product, we are using the organism Saccharomyces cerevisiae, a type of yeast. In its diploid form, this yeast has the inherent capability to undergo sporulation. This process, while natural, poses a risk, even in a controlled laboratory setting, because it could potentially allow the organism to escape into the external environment. Furthermore, it is worth noting that while Saccharomyces cerevisiae is generally benign, it carries the potential to infect those with compromised immune systems. Such infections can manifest in the lungs, leading to serious pulmonary conditions. Given these potential hazards, we re-evaluated and adjusted our research methodologies. To begin with, we strategically opted to use a haploid strain of Saccharomyces cerevisiae, which significantly reduces the risk of sporulation, thereby addressing one of the major concerns. Beyond this specific strain selection, we are also amplifying our efforts to maintain a pristine laboratory environment. By rigorously adhering to standard safety laboratory procedures that emphasize utmost cleanliness and precision, we are confident in our ability to curtail any possibility of spore contamination, ensuring both the safety of our staff and the surrounding environment.

Modified version AtBAG6

For inducing cell lysis in our yeast, we are using a modified version of AtBAG6, originally sourced from A. thaliana. This particular gene has the inherent capability to induce regulated cell death. There is a significant concern that, if not properly expressed, it might cause uncontrolled cell death, especially in the yeast S. cerevisiae. The intricate inclusion of the IQ motif is of particular interest as it plays a critical role in facilitating Ca²⁺-independent complex formation with CaM. This process is known to initiate programmed cell death, not just in yeasts but also in plants. However, it is worth noting that for other organisms, particularly humans, the gene remains benign and poses no known threat. To navigate the potential hazards, several safety measures have been put in place. A primary strategy is the use of specific promoters designed to induce apoptosis, but only in S. cerevisiae. The vector chosen for this purpose integrates exclusively at the HO locus site in S. cerevisiae and can also be found within E. coli, serving as a location for its replication and storage. Recognizing the gravity of potential contamination, we have implemented sterile procedures. This includes the consistent autoclaving of all equipment, ensuring that all potential contaminants are eradicated. A dedicated set of tools is reserved solely for work related to S. cerevisiae, eliminating any potential for cross-contamination. Furthermore, to maintain the highest level of safety and precision, our laboratory refrains from conducting concurrent work on multiple organisms, ensuring that focus remains and risks are minimized.

Cucurbita pepo

Lastly, we also need to address the plant we are testing Fungilyzer on. It is Cucurbita pepo, more commonly known as zucchini. This plant, while beneficial in many respects, comes with certain inherent vulnerabilities. One significant concern is its susceptibility to insect activity, particularly the laying of eggs on the plant. Moreover, the pollen produced by Cucurbita pepo can be a source of discomfort for some people, as it has the potential to induce allergic reactions in those who are predisposed. If not monitored closely, there is a risk of unintentional release of Saccharomyces cerevisiae into the environment via the runoff created by watering the plants.

Given these challenges, significant reconsiderations and enhancements have been implemented in our research approach. One of the foremost countermeasures introduced is the incorporation of a protective sand layer during the planting phase. This layer acts as a deterrent to insects, significantly reducing their activity around the plant. To tackle pollen-related concerns, our laboratory is equipped with a state-of-the-art ventilation system. This system is designed to ensure minimal retention of pollen within the laboratory environment. In addition, we have taken proactive measures to educate our team members about the potential presence of pollen, ensuring they are prepared and vigilant. To address the water-related risks, we have instituted a protocol where water from the plants undergoes detailed phosphate testing. Post-testing, the water is subjected to autoclaving to ascertain its sterility. Additionally, we spared no effort when it comes to sterilization. Every piece of equipment, material, and even the soil in which the plants grow undergo comprehensive sterilization processes. To further alleviate concerns regarding the interaction with S. cerevisiae, the specific strain we employ is not able to form spores, as mentioned earlier. Lastly, the soil chosen for planting has been selected for its near-sterile properties, further enhancing our risk minimization efforts.

Conclusion

In conclusion, the potential rewards of our finalized Fungilyzer are promising, and we recognize the importance of rigorous safety precautions. By addressing the outlined concerns, we aim to ensure that our product benefits both the environment and humanity, minimizing risks. With our detailed and thorough measures in place, we are confident that our research can be conducted safely and effectively. Prioritizing human and environmental safety at every juncture, Fungilyzer stands poised to not only meet but potentially surpass its ultimate goals, achieving enhanced agricultural yields and promoting sustainable nutrient usage, all for the betterment of our global food systems.