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Biosafety refers to the potential risks of unintended exposure or incidental release of biological agents to humans and the environment [1]. Addressing these issues is especially important in the case of our project, as after introducing a living organism into the soil the possibilities to control its behaviour are restricted to the genetic circuits that were implemented prior to its release. Thus, to minimise the potential risks we have followed a safe-by-design approach that would provide the user with the necessary controls for proper deployment and biocontainment.

For these reasons, not only did we seek to construct a couple of kill switch systems, but also to limit the production and secretion of antiflorigens to a specific set of conditions. In addition to these, we barcoded our modified bacterium and designed a LAMP detection assay to guarantee the traceability of the bacteria upon release. This ensures a mechanism of quantification of the bacterium in the soil, for proper evaluation of both successful colonisation and containment efforts.


Biocontainment of our bacteria is ensured by making their survival dependent on the presence of cuminic acid, a substrate that will be externally delivered to the site of application. Only upon detection of this compound will the bacteria be able to express dapB, an essential gene that plays a key role in the production of lysine and diaminopimelate (DAP). This dependency is achieved by knocking dapB out of the genome and placing it on a plasmid under the control of an inducible system.

Antiflorigen expression is context-specific

The production and secretion of antiflorigen are possible only in close proximity to roots and after a sufficiently large colony has formed. While this adds another biosafety layer, it also enhances the survivability of our strain. Context-specific antiflorigen secretion ensures that this protein is delivered only to our designated target (the fruit tree), limiting its effect on other living organisms.

LAMP detection assay ensures certainty of containment (CoC)

Enabling farmers and regulatory authorities to easily detect the presence or absence of our genetically modified organism in the soil is one of the key features of PseuPomona and its application. Therefore, we set out to barcode our bacterium and develop a Loop-mediated isothermal amplification (LAMP) assay specific to this knocked-in sequence. Besides improving CoC, identification of a GMO’s barcode would support easy acquisition of publicly available containment measures [2]. For farmers, this tool would allow assessing whether colonisation of the applied GMO product was successful or not, thus reducing the false sense of protection of the crops when colonisation fails. Moreover, after the risk of late-spring frosts no longer exists and the bacteria are to be removed this assay would provide confirmation of their correct eradication.


Biosecurity aims to safeguard the well-being of both the environment and society from toxins, pathogens, and biological materials of perpetuate harmful consequences inside and outside of the biology research environment. Therefore, biosecurity prevention measures aim to impede their loss, diversion, theft, or intentional release [3]. Whether in the design, development, or production phases of engineering bacteria, it is thereby crucial to continuously contemplate and implement safe-by-design biosecurity precautions, whenever possible [3].

Thus, in order to mitigate misuse of our genetically modified microorganism (GMM), it was also necessary to consider the possible dual-use of our invention. These concerns often arise as scientific advancements, which initially advocated improving aspects of society, human health, or the environment, can also pose risks with the unpredictability of external malevolent actors [4]. Throughout the last decades, the knowledge and methods of synthetic biology have quickly expanded and thus, misuse of technologies for bioterrorism opportunities has become more tangible, reinforcing these concerns [4].

PseuPomona and biorisk

Biosecurity risks associated with PseuPomona can be focused on acknowledging that our bacterium comprises a delivery vector for proteins of interest into trees. This is where the dual-use possibility of our bacterium exists, as the deliberate expression and secretion of the antiflorigen in some conditions (e.g. later in the season), could harm food production. The antiflorigen gene could also be substituted by another gene, for example one that is toxic to the tree’s development. This is why implementing measures enabling the control and detection of a GMM is essential. Additionally, this would also allow the regulatory bodies to control the presence of our bacterium.

Moreover, horizontal gene transfer (HGT) poses another concern regarding the biorisk associated with GMMs [5]. However, in the eventuality of HGT of the antiflorigen cassette, it would only pose a biorisk hazard if the receiving bacteria possess Type III secretion system (T3SS), and are in the environment where both inducers necessary to activate the toehold are present.


From the start of PseuPomona’s development, we sought to implement safety-by-design measures whenever possible. Following this approach helps to minimise evermore complex biocontainment, biosecurity, and regulatory efforts that could hinder the future progression of such a project [6]. The safety-by-design approach can range from the choice of the bacterial chassis to the assembly of genetic pathways that establish a safe regulatory hierarchy. Our project addresses these aspects in the following way:

Safe chassis - Pseudomonas fluorescens SBW25 is a non-pathogenic bacterium commonly found in soil and is classified as a plant-growth-promoting rhizobacterium (PGPR) [7]. The microorganisms included in this group are known for interacting with many plant species for instance by secreting beneficial compounds or fixing nitrogen. Consequently, PGPRs can significantly mitigate the effects of abiotic stress such as high salinity, pollution, and drought [6]. Due to these characteristics, many PGPR species including P. fluorescens, are used in commercially available biofertilizers or biostimulants. Several genetically modified P. fluorescens bacteria have also been approved for environmental release for a variety of uses, being considered a safe vector [8]. Due to these characteristics and the genetic availability of this bacterium, our project used P. fluorescens SBW25 as its chassis of choice.

Secretion of antiflorigens - The toxicity of antiflorigens is not known as they are not studied as heterologously expressed proteins. Nevertheless, since these proteins in varying concentrations are native to plant species, an assumption can be made that - at least for plants - antiflorigens are non-toxic [9]. Therefore, leaky expression of small amounts of the antiflorigen protein is expected not to interfere with the natural flowering process. Potential toxicity to other elements of the ecosystem such as the native microbial community is not expected. However, it has to be studied prior to the release of our bacteria into the environment.

Biocontainment - Introduction of synthetic genetic circuits allowing to control the behaviour of bacteria upon environmental release adds another safety layer. As such, our bacteria will be equipped with mechanisms that keep them alive in the presence of cuminic acid and induce cell death in the presence of rhamnose. Additionally, the LAMP detection assay helps to ensure the correct eradication of the bacteria.

In addition to these, we designed the experiments in a way to prevent the pairing of modified P. fluorescens SBW25 with Arabidopsis thaliana Col-0. Together, A. thaliana Col-0 would also be considered a GMO, which would result in the necessity of working in an ML-2 laboratory. To achieve this, the experiments addressing the bacterium’s ability to secrete antiflorigens and their phenotypical effects in plants were conducted separately.

Lab safety

Safety is an important part of any research environment and for the success of PseuPomona it was no different. Thus, while pursuing the development of a safe engineered bacterium, we also wanted to ensure that its development was executed in a safe manner. Laboratories often contain significant risks for the workers, such as extreme temperatures, dangerous chemicals, and biohazards. In addition, genetically modified organisms could escape from the laboratory, potentially causing damage to the environment. Therefore, it is imperative that lab workers follow safety rules that help manage the risks associated with lab work.

Our team followed two lab safety tours in preparation of the used facilities for the conduction of experiments. During these, we were educated on the safe handling of chemicals, microorganisms, equipment, biological waste containment, emergency situations, lab safety rules and other practical aspects. Afterwards, we were required to pass an official lab safety examination before the start of any experiment.

Agata in an ML-1 laboratory.

During the PseuPomona project, we worked in ML-1 laboratory facilities (equal to Biosafety level 1). The classification of these facilities is based on the amount of containment necessary to minimize the risks associated with the manipulation of a given microorganism’s risk classification group [10]. To understand the correct settings for our experiments, this aspect was discussed with several lab personnel, including technicians, and meetings with biosafety officers of Wageningen University & Research.


Apart from considering only the more practical biosafety and biosecurity measures, ethics should always be an integral aspect of synthetic biology projects. Despite implementing all the measures minimising the risks associated with releasing a GMO into the environment, still, many ethical questions arose throughout the project.

The main ethical concerns focused on the traceability of the bacterium after its release to the environment and the outcome of phenotypical changes in plants following the uptake of antiflorigens. Other ethical questions wonder whether the introduction of our bacterium would lead to microbiological or chemical changes in the soil and the economic accessibility of this tool. In addition, concerns regarding the future approval possibility of such a product would vary between regions also raising questions about geographic accessibility.

In addition to addressing some of these by implementing safe-by-design strategies, we also sought to gain and share knowledge on the ethics of applied synthetic biology with others throughout our education outreach and communication journey.

Although we recognise we do not have answers to all the questions, various moments during PseuPomona’s development improved our ability to highlight and questions ethical issues associated with our project. The most relevant ones are enumerated below:

  • Meeting with Dr. Cécile van der Vlugt-Bergmans, GMO Risk Assessor at the GMO office of the RIVM (Dutch National Institute for Public Health and the Environment)
  • Ethical discussions with Dr. Zoë Robaey, Assistant Professor in Ethics of Technology and Safety Officer Steven Aalvin at Wageningen University & Research.
  • A panel discussion organised by us, titled “The Role of Synthetic Biology in Agriculture”, in which one of the panellists was Alessio Gerola, a PhD candidate in Ethics of Technology at the Philosophy Group of Wageningen University

[1] Beeckman, D. S. A., & Rüdelsheim, P. (2020). Biosafety and Biosecurity in Containment: A Regulatory Overview. Frontiers in Bioengineering and Biotechnology, 8.

[2] De Lorenzo, V., Krasnogor, N., & Schmidt, M. (2021). For the sake of the Bioeconomy: define what a Synthetic Biology Chassis is! New Biotechnology, 60, 44-51.

[3] Hoffmann, S. A., Diggans, J., Densmore, D., Dai, J., Knight, T., LeProust, E., Boeke, J. D., Wheeler, N. E., & Cai, Y. (2023). Safety by design: Biosafety and biosecurity in the age of synthetic genomics. iScience, 26(3), 106165.

[4] Miller, S., & Selgelid, M. J. (2007). Ethical and philosophical consideration of the dual-use dilemma in the biological sciences. Science and Engineering Ethics, 13(4), 523–580.

[5] Getachew, A., & Andualem, B. (2020). Biosafety issues of unintended horizontal transfer of recombinant DNA. In IntechOpen eBooks.

[6] Mohanty, P., Singh, P. K., Chakraborty, D., Mishra, S., & Pattnaik, R. (2021). Insight into the role of PGPR in Sustainable agriculture and environment. Frontiers in Sustainable Food Systems, 5.

[7] David, B. V., Chandrasehar, G., & Selvam, P. N. (2018). Pseudomonas fluorescens : A Plant-Growth-Promoting Rhizobacterium (PGPR) With Potential Role in Biocontrol of Pests of Crops. In Elsevier eBooks (pp. 221–243).

[8] Wozniak, C. A., McClung, G., Gagliardi, J. V., Segal, M., & Matthews, K. (2012). Regulation of genetically engineered microorganisms under FIFRA, FFDCA and TSCA. In Springer eBooks (pp. 57–94).

[9] Higuchi, Y. (2018). Florigen and anti-florigen: flowering regulation in horticultural crops. Breeding Science, 68(1), 109–118.

[10] WHO, “Laboratory biosafety manual Third edition World Health Organization,” World Heal. Organ., pp. 1-178, 2004

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