Safety and Security

Introduction

All scientific research raises questions of safety and security. While working with organisms that have been genetically modified, strict adhesion to biosafety protocols is required. This to protect researchers in the laboratory and to prevent accidental release of the organism into the environment. Beyond safety, it is equally important to address biosecurity, acknowledging that once research is made public, it may be misused by individuals with ill intentions. These concerns must also be placed within the legal and regulatory frameworks specific to each region, ensuring that the work aligns with established standards and safeguards.

Biosafety

Biosafety includes all the safety measures taken in the lab including facilities, choice of chemicals and biological agents used, and training to avoid the release of organisms from the lab.

Lab Safety

Before the start of experiments, all team members assigned to work in the lab were required to attend health, safety and environment training provided by Frédérick Coosemans. The session included topics such as waste disposal, emergency procedures, fire safety, chemical spill response and the location of safety equipment including emergency kits, showers, fire extinguishers within the lab. Our wet lab activities took place in the lab of our principal investigator (PI) Vitor Pinheiro, located in the Rega Institute of KU Leuven. Furthermore, some procedures were consistently followed in the lab. Personal protective equipment was always worn, and surfaces were disinfected regularly and kept well-organized by labelling. In addition, thorough lab cleaning was carried out weekly. During our cell culture experiments a biosafety cabinet was used and at the end of each experiment, UV light was used to sterilize the cabinet. Waste management was also carried out in accordance with instructions given by the lab responsible and the PhD students working in the lab. These are further described below:

• Personal Protective Equipment (PPE): all members working in the laboratory wore PPE. This includes lab coats, gloves and safety goggles.
• Lab cleaning and sterilization: surfaces in the laboratory were disinfected regularly with ethanol. A deep cleaning of the lab was also performed weekly, to ensure proper cleanliness of all equipment used.
• Use of lab space: experiments with Escherichia coli, Bacillus subtilis and Phytophthora strains were performed on designated work benches and in biosafety cabinets. These biosafety cabinets were kept sterile using two procedures. Disinfection with ethanol and use of UV at the end of each experiment. We used 30 minutes of UV sterilization after working with B. subtilis and E coli. While for Phytophthora strains we have used 40 minutes of UV treatment post-experiments, to ensure that there was no risk of pathogen contamination.
• Autoclaving all media and pipette tips used during the experiments were autoclaved prior to use.
• Labelling: all experimental materials such as petri dishes and test tubes were labelled with the date of the experiment, the name of the member who had prepared them and their respective content. This was done for safety reasons to keep the lab organized and to avoid spore formation.
• Waste management: waste produced during the experiments was disposed of in appropriate waste containers. Any objects that came into contact with micro-organisms, such as petri dishes and pipette tips, were first sterilized through irradiation and then disposed of in the designated waste containers.

Biological Safety

During our experiments, we used E. coli (DH10β) and B. Subtilis 168. Both of these strains are classified as BSL-1 organisms, meaning they pose little to no threat of infection. We also used P. capsici and P. palmivora, which are BSL-1 organisms too. Since our work was conducted in a BSL-2 laboratory, the appropriate safety measures and procedures were in place to allow these experiments to be carried out safely.

Containment Strategy

B. subtilis, the workhorse of recombinant protein production on an industrial scale, is regarded as a GRAS microorganism [1]. Despite its established status, we are aware of the potentially adverse consequences arising from the escape of our biocontrol agent into the environment. Hence, to prevent the risk of horizontal gene transfer, disruption of the microbiome and human life endangerment, we have developed a theoretical model of a kill switch. This system will ensure biocontainment by promoting the conditional death of the bacterial cells upon release from the non-target environment.

The primary layer in our kill switch design is inspired by the Demon and Angel essential gene construct developed by Kato & Mori [2]. The power of this system lies in its ability to address and diminish the intrinsic genetic instability of synthetic gene cassettes introduced into a microbial chassis. Engineering a microbe to carry additional genes can impose a severe metabolic burden and reduce the fitness of the organism, ultimately driving formation of loss-of-function mutations. The Demon and Angel expression construct of an essential gene is carried on a plasmid with the chromosomal counterpart being deleted. It then tackles the issue of escape mutants by allowing basal expression of the gene to facilitate host viability, but triggers death of the cell when the same essential gene is overexpressed. With this mechanism underlying the primary layer of our kill switch, we attempted to formulate a Demon and Angel genetic circuit with the tyrosyl-tRNA synthetase essential gene regulated by a theophylline-riboswitch. While the tyrS gene was selected in the original study, genetic screening methods must be implemented to identify candidate essential genes in B. subtilis.

Theobromine is an alkaloid that is produced in high concentrations by cocoa plants, especially in the pods and the leaves [3]. While not many regulatory gene elements responsive to theobromine have been characterized, synthetic riboswitches reactive to theophylline, another alkaloid with high structural similarity to theobromine, have been developed in recent years [1], [4]. When combined with the strong constitutively expressed promoter P43 in B. subtilis [5], a dose-dependent induction of expression by theophylline was evinced, with implicated applications in overproduction of proteins.

We postulate that this riboswitch design could be leveraged and adapted to theobromine by computational modelling of the aptamer domain. By doing so, we aim to engineer a repressive riboswitch responsive to theobromine for implementation in the design of a kill switch for our project. Ideally, in the presence of theobromine, a conformational change in the riboswitch should be triggered which would then result in the formation of a terminator hairpin that partially or weakly blocks transcription [6], thereby allowing only for low level expression of the essential gene, such as tyrS as demonstrated in E. coli, under the control of the constitutive promoter P43. But in the absence of theobromine, the riboswitch must fold on binding to its ligand to release the terminator hairpin and form an anti-terminator hairpin, thereby promoting transcription and overexpression of the essential gene, leading to cell death (Figure 1).

Figure 1. Theobromine-riboswitch regulation of essential gene tyrS expression as part of Demon and Angel kill switch.

To make our biocontainment strategy more robust in nature [7], the primary layer of our kill switch will be supplemented by a secondary layer, an inducer-dependent toxin-antitoxin system [8]. Xylose was selected as the inducer, as this is a sugar that is predominantly found in the walls of plant cells. In intact cell walls, xylose constitutes hemicellulose that reinforces the structure of the wall. But on degradation by agents such as cell wall degrading enzymes secreted by plant pathogens, such as Phytophthora, xylose is released in its unbound form [9].
Hence, xylose will serve as an environmental input for a synthetic gene construct comprising the xylose-controlled operon integrated into the chromosome of the chassis. This will include the PxylA promoter and XylR repressor, the gene for the TetR repressor under the control of the PxylA promoter, the MazF toxin gene under the control of a TetR repressible promoter (Ptet) and the MazE antitoxin gene under the control of a strong constitutive promoter such as Psdp. In the absence of xylose, the promoter PxylA will not be activated (due to repression by XylR repressor). As a result, the TetR repressor gene will not be expressed, and hence the Ptet promoter of the MazF toxin gene will not be subjected to repression, thereby enabling the production of the MazF toxin in higher quantities than the MazE antitoxin.
This is the ideal scenario expected to occur in the case of escape of the biocontrol agent from the non-target environment where xylose availability in its free form is expected to be limited. But when xylose is present, as in the case of infection of the cocoa plant by Phytophthora, the PxylA promoter will be activated and the TetR repressor gene will be expressed. This should cause the repression of the toxin gene expression. To maintain stability in the system and reduce the risk of premature or unintended killing of the cell, the antitoxin gene is constitutively expressed to sequester toxin production in the case of leaky expression. Prior to field-level implementation, both riboswitch-modulated and xylose-regulated kill switch must be probed and validated to determine parameters such as dynamic range, kinetics and activation thresholds of the genetic circuits.

Figure 2. Xylose induction of MazF toxin – MazE antitoxin kill switch.

Biosecurity

Biosecurity includes principles and practices designed to prevent unauthorized and potential misuse of biological research, organisms or information, whether through theft or intentionally harmful use. We therefore needed to assess if the project being developed was at risk for any of this to happen.

Risk Assessment

The risks were assessed with the aid of the WHO laboratory biosecurity guidance [10].

• Physical security risks: risks that are directly linked with the laboratory used during the experimental phase of the project. They include access to the laboratory to unauthorized individuals, improper use of equipment or theft of equipment.
• Biological security risks: risks that are associated with the biological agents used in the laboratory. These include the loss, theft or release of biological material.
• Personnel security risks: this refers to risks associated with individuals involved in the project, including potential hazards posed by the experiments to the group members, as well as risks arising from their non-compliance.
• Information security risks: risks related to project data, including methods and results generated during the research. They encompass unauthorized access, espionage or misuse of sensitive information.

Risk Management

After the identification of each risk, measures were taken to minimize their risk and prevent a biosecurity problem.

• Physical security risks: The risk of unauthorized access and theft of equipment was minimized by the strict pre-existing access policy in the Rega Institute. Only authorized personnel were able to enter the building. The safety training and the training provided by PhD students during the experimental phase also prevented the improper use of equipment.
• Biological security risks: All biological strains used in the laboratory were classified as BSL-1, indicating a low security risk. Furthermore, mandatory training and restricted access for unauthorized personnel significantly reduced the risk of theft of biological materials. Regular inventory checks were also conducted throughout the experimental phase to ensure proper monitoring and control to avoid the loss of any material.
• Personnel security risks: The strains and chemicals used in the lab were chosen to ensure minimal risk to team members. When hazardous chemicals were used, they were handled strictly following all security procedures mandated by the university. Throughout the duration of the project, no instances of non-compliance were observed as multiple people were always in the lab together under the guidance of PhD students.
• Information security risks: Project data was handled with care during the duration of the entirety of the project. All of the data was stored in platforms and clouds with security measures that prevented the possibility of anyone unauthorized accessing sensitive information. Additionally, any sensitive information that may arise during the project will be included in the supplementary materials section, thus limiting its availability.

Additionally, another risk that has become increasingly important in the last years is antimicrobial resistance. Antimicrobial peptides are less likely to induce bacterial resistance compared to other types of antibiotics, as they primarily target microbial membranes. Consequently, their use does not contribute to antimicrobial resistance as much as other solutions and would minimize it becoming a bigger problem in the future [11].

Legal Framework

The use of genetically modified organisms is strictly regulated under both European Union law and the legal frameworks of cacao-producing countries. These regulations are designed to protect human health, biodiversity, and make sure that biotechnological innovations are developed responsibly. It is therefore essential to understand them for potential future trials.

International Legal Frameworks

International legal landscapes provide the foundation for national biosafety regulations. The most relevant international agreement concerning GMOs is the Cartagena Protocol on Biosafety [12], which regulates the safe handling, transport and use of living modified organisms. It emphasizes risk assessment and management by taking a precautionary approach to the subject. Its primary goal is to protect biodiversity from the possible risks of GMOs.

European Union Framework

Within the EU, the framework governing the release of GMOs into the environment is the Directive 2001/18/EC [13]. For an agricultural application, approval would require submitting a full environmental risk assessment to the European Food Safety Authority (EFSA). This includes data on potential effects on the soil, on other organisms present in the environment and the possibility of horizontal gene transfer. Additionally, public participation by citizens is also made possible which means an increased scrutiny of the project, making the process lengthier. Finally, the authorization of the bacteria as a bio-control agent by the EU would allow the release of the GMO for specific application.

Cacao-Producing Countries Framework

Our product would be commercially available in countries with cacao-producing capabilities, this means that their legal structure is especially important for our product. We looked across four of the biggest cacao producers in three different continents to understand the different approaches, each of them was taken into consideration to tackle the issue of biosafety and how that could affect PhytoBlock. It is also important to note that all the countries mentioned below have signed the Cartagena Protocol on Biosafety, meaning they have all taken a precautionary approach to biotechnology.

• Ghana: As one of the largest cacao producers, Ghana has developed the Biosafety Act, 2011 (Act 831) [14]. It establishes the National Biosafety Authority, which is responsible for overseeing GMO approvals, conducting risk assessments and facilitating public engagement. Moreover, to operationalize this law, Ghana introduced the Biosafety (Management of Biotechnology) Regulations, 2019 (L.I.2383), which provides detailed procedures for its implementation [15]. Under this system, PhytoBlock would be classified as an “introduction into the environment”, requiring prior written authorization from the National Biosafety Authority before commercialization.
• Côte d'Ivoire: As the largest cacao producer in the world, the regulation of Côte d'Ivoire is especially important for the project. In 2016, the country adopted the law No. 2016-533 which provides the legal basis for the safe use of biotechnology [16]. It establishes requirements for the import, export and release of GMOs. The National Biosecurity and Biosafety Commission (CNBIOS) and the National Biosecurity Observatory (ONBIOS) oversee the implementation of the law. [17]
• Indonesia: The primary legal tool of Indonesia is Regulation No. 21/2005 on the Biosafety of Genetically Engineered Products, which establishes the framework for GMO oversight. Under this regulation, two key institutions are responsible for managing GMO research and environmental release. The Commission for Biological Safety of Genetic Engineering Products (KKH) issues certificate of biosafety, while the Technical Team for Biological Safety (TTKH) conducts the relevant technical evaluations. Market introduction of GMO products into the country includes several steps involving these two bodies. It requires comprehensive risk assessments and compliance with strict biosafety standards before authorization is granted, making the process lengthy but structured [18], [19].
• Ecuador: Ecuador is the fourth largest producer of cacao in the world. It has restrictive regulation based on Article 401 of the 2008 Constitution, which prohibits the introduction of GMOs into the country unless the proposal is approved by the President and the National Assembly. While the Organic Code of the Environment now provides exemptions based on case-on-case basis, approvals remain extremely rare and a major policy shift is needed for any field application of GMOs [20], [21]. A project involving the release of a genetically modified microorganisms, would face significant legal and political obstacles with approval unlikely under the current policy environment.

The regulatory environments of major cacao-producing nations present both opportunities and challenges for the commercialization of PhytoBlock. Ghana and Indonesia have established biosafety frameworks aligned with the Cartagena Protocol, offering structured though rigorous pathways for approval. In contrast, Côte d'Ivoire's system remains largely non-operational, resulting in an effective prohibition on GMO use, while Ecuador maintains a constitutional ban with only rare exemptions, making approval highly unlikely. Overall, this suggests that while commercialization may be viable in Ghana and Indonesia under strict compliance with biosafety requirements, introduction in Côte d'Ivoire and Ecuador would be far more difficult due to legal and political constraints in place.

Our project is designed for use by farmers in cocoa-producing countries. During our conversations with farmers, we found them open to applying our solution on their small-scale farms. This made it our responsibility to ensure that our approach is not only environmentally safe but also safe for the farmers themselves. Since our biocontrol strategy relies on antimicrobial peptides (AMPs) that are non-hemolytic and therefore harmless to human health, we do not foresee any risks to the farmers using our product.

In terms of biosecurity, it is important to reflect on the concept of dual use research of concern (DURC). While our project does not fall under DURC as defined by international guidelines, we recognize that existing frameworks such as the Biological Weapons Convention mainly address human and animal pathogens [22]. We suggest that future DURC discussions also consider agricultural biotechnology, since many iGEM projects are based on food and crop-related challenges. Therefore, unintended consequences such as the development of pathogen resistance or horizontal gene transfer could lead to significant agricultural and environmental impacts, ultimately affecting human well-being.