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.

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.