Safety

~Overview~

If the iGEM way of work taught us one thing, it is to plan ahead and always have a backup plan. As a result, safety became a value we learned to cherish and always put into effect as we develop into the scientists of tomorrow. The proposal of an engineered bacterial population on our part also meant that we had to strictly follow all safety measures, both within and outside the lab, as the project’s future implementation indicated the need for robust biocontainment measures for our product.

~Biosafety~

As the World Health Organization (WHO) suggests;
“Biosafety is the safe working practices associated with the handling of biological materials, particularly infectious agents.” [1]

As a result, biosafety practices include containment principles and technologies able to prevent unintentional exposure to pathogens and their respective dangerous products or unintentional release of those pathogens. As such, in-laboratory practices have evolved to include stringent control of biological materials used.

Lab safety

Since we were working with organisms in a lab setting, we were thoroughly trained in the proper use of equipment (such as using the biosafety cabinet, working under the flame, surface cleaning and disinfection, using a chemical fume hood), proper attire (such as wearing lab coats, gloves and double gloves where needed eye protection) and proper waste disposal (such as use of the autoclave, separation of waste into different categories). Additionally, throughout our experimental phase, we were always supported whenever any questions arose. We were always under close supervision for the duration of our experimental procedures, to minimize risks and ensure the best possible outcome of our experiments.

Microorganisms

For our experiments, we have used the following bacterial strains;

Bacterial Strain	Provided by
E. coli  DH5α	iGEM Thessaly 2023 stock
E. coli  BL21 (DE3)	iGEM Thessaly 2023 stock
E. coli  PIR2	Dr. Esteban Martínez-García, CNB-CSIC
E. coli  DH5α-λpir	Dr. Esteban Martínez-García, CNB-CSIC
E. coli  HB101	Dr. Esteban Martínez-García, CNB-CSIC
P. putida  KT2440	Dr. Esteban Martínez-García, CNB-CSIC
P. putida  KT2440 Δrnc	Dr. Esteban Martínez-García, CNB-CSIC

Table 1: Bacterial strains used by iGEM Thessaly 2024.

All strains kindly provided by Dr. Esteban Martínez-García, were used for the chromosome insertion for parts of our design, following information provided in Zobel et al. (2015) [2]. For more information on our design, head over to our Project Design and Experiments pages.

E. coli strains

E. coli DH5a was used for the clonings. E. coli BL21 (DE3) was used in promoter characterization and engineering experiments, for the expression of sfGFP. E. coli PIR2 (p Tn7- M), E. coli DH5α-λpir (pTnS-2), E. coli HB101 (pRK600) were used for the integration of some of our constructs into the chromosome, we prepared by having extensive meetings with Dr. Esteban Martínez-García, an author of the original publication that suggested the way of integration we decided upon. [2] Our instructor, Dr. Maria Tsampika Manoli also made sure that we understood the risks and the key steps in these protocols. Both of them also provided us with information regarding the safe handling and disposal of these microorganisms.

P. putida strains

P. putida KT2440 was used to integrate some of our constructs into its chromosome. P. putida KT2440 Δrnc was used in promoter characterization and engineering experiments, for the expression of sfGFP. For the handling of the P. putida strains, both Dr. Esteban Martínez-García and Dr. Maria Tsampika Manoli took great care in ensuring our understanding of handling and safe disposal. Dr. Manoli served as our instructor, with a specialization on our selected chassis, as she has expertise in the utilization of P. putida. Dr. Manoli supervised our work through weekly meetings, to ensure high standards of work.

Verticillium dahliae

The fungus Verticillium dahliae, a plant pathogen, was also used. Though not dangerous to humans, an allergic reaction due to inhalation of its components could potentially happen. It can, however, be avoided by the utilization of proper practices. Since it is classified as a Risk Group 2 organism, due to its potential to cause harm to plant life, we made sure that all members of the Wetlab team were trained on its handling. Any sort of handling of the fungus was performed in a designated Biosafety Class II Cabinet, to maintain sterile conditions for our specimen, but also to prevent the spreading of any sort of contaminants and/or spores of the fungus itself. This is achieved through the use of a HEPA filter, equipped to filter the air so as not to have any spore dispersal. Any handling of the fungus was done in a contained environment, with the use of gloves and lab coats being a constant in our practices. The use of a Risk Group 2 organism meant that we had to be familiarized quickly and extensively with high standards of care, for the protection of our specimen, of our lab mates and of our environment. The Verticillium dahliae strain for our experiments was provided by the Laboratory of Plant & Environmental Biotechnology of the Department of Biochemistry and Biotechnology, University of Thessaly. Our handling of it was always supervised by a specialist in the same lab. Also, our instructor Afrodite Katsaouni, PhD Candidate supervised weekly our process to ensure high standards of work, as she is familiar with the handling of Verticillium dahliae. We also made sure to discard all microorganisms properly, to maintain safety compliance. Verticillium dahliae, E. coli, and P. putida were checked in using the iGEM provided form.

Hazardous reagents and procedures

All hazardous reagents were handled with great attention. Double gloves, long lab coats, and glasses were always present in the handling of these chemicals. For our experiments, we used chemicals such as:

- Ethidium Bromide: for the electrophoresis. We were trained to recognize the designated areas for its use to avoid skin contact, as it is a mutagen. We were also extensively tutored in the making of the gel to avoid any possibility of inhalation of this substance.

- TRItidy G™: exclusively used in a chemical hood for RNA extraction, essentially avoiding inhalation of harmful substances for people and the environment. Used under supervision and dressed accordingly - safety glasses, lab coats, double gloves for our protection.

- Phenol - chloroform: exclusively used in a chemical hood for the dsRNA production, essentially avoiding inhalation of harmful substances for people and the environment.

For our experiments, we faced hazardous procedures such as:

- Tissuelyser II: for our fungal tissue samples. Used under supervision and dressed accordingly - safety glasses, lab coats, gloves for our protection.

- UV Light use for electrophoresis: We were trained to safely utilize the UV Light necessary for the visualization of our electrophoresis results.

We also made sure to discard all chemicals properly, in order to maintain safety compliance.

Parts

1.Plasmids

The Golden Braid vectors pUPD2, pDGB3alpha 1, pDGB3alpha2, and pDBG3ω, were recovered from the iGEM Thessaly 2023 glycerol stock.

The Golden Standard pSEVA23g19 [g1], pSEVA23g19 [g2], pSEVA63g19 [gE], and pSEVA63g19 [gA] plasmids we used were kindly provided to iGEM Thessaly 2023 by Dr. Blas Blazquez, Researcher at CNB-CSIC. The recovery was done according to the instructions provided on the Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, CNB-CSIC website.

The pTn-7M, pTnS-2, pRK600 plasmids were isolated by us from the strains sent to us by Dr. Esteban Martínez-García, Researcher at CNB-CSIC, mentioned in Table 1 above.

The pSEVA23g19 [g1], pSEVA23g19 [g2], pSEVA63g19 [gA], pTn7-M, pTnS-2, pRK600 plasmids were checked in using the iGEM provided form, as they contained an origin of transfer (oriT) site.

2.Parts

All parts were synthesized by International DNA Technologies (IDT), as part of the sponsorship provided by the competition. All primers were synthesized by Eurofins Genomics. You can refer to our Part Collection page for an in-depth analysis of any parts used.

~Biosecurity~

As mentioned in Huber et al. (2022) [3];

“A biosecurity measure (BSM) – is the implementation of a segregation, hygiene, or management procedure (excluding medically effective feed additives and preventive/curative treatment of animals) that specifically aims at reducing the probability of the introduction, establishment, survival, or spread of any potential pathogen to, within, or from a farm, operation or geographical area.”

For us, that meant that we should implement the principles of biosecurity at the core of our design to achieve the safeguarding of this novel bmRNAi technology by choosing a nonpathogenic chassis, ensuring that our system does not lead to RNAi off-targets and by working with not harmful parts and safe materials for proof of concept.

Design Safety

The proposed bmRNAi raised concerns, as it could potentially be misused against humans to silence essential genes. Our team understands this and is dedicated to preserving the protection of human health from any harm posed by this technology. The staff of the Molecular Biology and Genomics Laboratory, Department of Biochemistry and Biotechnology, University of Thessaly, were tasked with our training regarding correct RNAi employment techniques. Furthermore, our Principal Investigator, Prof. Kostantinos Mathiopoulos, a professor of Molecular Biology and expert in RNAi, made sure to inform us of the importance of RNAi technology and its proper implementation in our practices, essentially granting us the necessary knowledge and passion to preserve biodiversity. This was especially helpful in our designing phase, as we steadily screened sequences through BLAST, in order to pinpoint species-specific loci in Verticillium dahliae genes. This led to the selection of low homology regions, practically limiting cross-species off-target effects.
As another measure, we have added the integration of the T7 polymerase into the bacterial chromosome, as it drives the activation of our dsRNA production. This way, we achieve the minimization of horizontal gene transfer risk. We, also, chose Pseudomonas Putida for our Design chassis, a Plant Growth Promoting Rhizobacteria (PGPR) that  has the ability to colonize olive roots, while it is considered a Level 1 Safety microorganism. [5] For more information on our design, head over to our Project Design Page. Finally, our experiments are entirely conducted in the laboratory, thus ensuring no contact of unsafe substances with the local outside environment. 

However, as we read and developed our project holistically, all departments came to a common realization; everyone on our team can play their part in order to truly encapsulate the essence of Safety in a biological system. After careful consideration, each department landed on the part that they could play in the materialization of this ideal.

Our Human Practices department, in collaboration with the Wetlab department, participated in the gathering of information from stakeholders and specialists; understanding the real-world implications of our project is vital. We have engaged with farmers to discuss current V. dahliae management methods and how our engineered bacterial formulation could be integrated into existing agricultural practices. This consultation helps us design a solution that is not only scientifically sound but also practical and acceptable for end-users. Feedback from these stakeholders has been invaluable in shaping our project’s goals and ensuring that our approach is feasible and safe for deployment in the field. In addition to our consultations with stakeholders, we have sought advice from recognized experts to refine our risk management strategies. For more information head over to our Integrated Human Practices page. 

Our Drylab department has chosen to use in silico simulations to predict how our genetically modified organisms (GMOs) will perform in open fields. This approach involves creating virtual scenarios to estimate how these organisms might interact with their environment, potential risks, and overall performance. Additionally, they have developed a hardware device for testing biocontainment measures in situ and monitoring bacteria-bacteria interactions. This hardware has the potential to enable the evaluation of containment strategies in practice, helping to ensure that GMOs remain controlled and do not negatively impact the environment.

This meant that all departments worked in tandem to achieve high standards of a holistic approach product that abides by guidelines regarding safety.

Figure 1: Collaboration of all of the iGEM Departments to create a safe Project Design

~Biocontainment~

As the Administration for Strategic Preparedness and Response of the U.S. Department of Health and Human Services underlines;

“Biocontainment is a component of biorisk management. The overall objective of biocontainment is to confine an infectious organism or toxin, thereby reducing the potential for exposure to laboratory workers or persons outside the laboratory, and the likelihood of accidental release to the environment. Physical containment is achieved through the use of laboratory practices, containment equipment, personal protective equipment, and laboratory and facility design.”[4]

As a team, we took biocontainment very seriously both during our experiments and when considering the implementation of our project and the risk of release of genetic material. During the time we spent in the lab, we always made sure to follow all the safety measures necessary to protect ourselves and other researchers (wearing proper attire, working in the designated laboratory spaces) and avoid any release of genetic material in the environment (proper waste disposal).
To ensure that our bacterial system won’t spread in the environment we first thought of a kill switch. Kill switches are genetic circuits designed to trigger bacterial cell death by activating the expression of a toxic gene in response to specific environmental conditions, like temperature or chemical compounds [6,7]. While there have been many attempts to develop reliable kill switches, there is a major disadvantage; they don’t last long. Because bacteria evolve and adapt rapidly, even in a span of a few days, random mutations can occur that disable the kill switch [8]. Another thing that Afrodite Katsaouni, Phd Candidate pointed out is that the leakiness of the promoter regulating the toxin gene would cause a lot of stress in the system. The bacteria, already confronted with the challenging environment of the soil, would have trouble growing.  Our device should be able to live in the plant root long enough to detect and fight the pathogen, so our biocontainment approach's longevity and reliance are critical.  So we decided on two approaches: 1) chemotaxis, where we have designed a gate that stops our bacteria from reproducing unless the gate is activated by the Ave1 protein from V. dahliae. This makes sure that the system only works when this specific pathogen is present.  2) double auxotrophy, where our system requires specific nutrients provided through our irrigation formula to survive and spread. This ensures that the engineered organisms can only thrive in environments where these conditions are met.

More specifically, we designed  a gate that activates the proliferation of the engineering bacteria in the presence of our target, V. dahliae. We designed a biological gate in our system by leveraging the Verticillium dahliae’s protein effector Ave1 as a key activator [9]. When Ave1 binds to the Ve1 receptor expressed in the engineering bacteria, it triggers a signaling cascade, as it is shown in Figure 2. The binding activates the EnvZ kinase, which transfers a phosphate group to the response regulator OmpR [10]. The phosphorylated OmpR then binds to the ompC promoter, leading to the production of DnaA, a key regulator for initiating the bacterial cell cycle because it is needed to start DNA replication. The ompC promoter replaces the DnaA1 promoter of the endogenous bacterial DnaA [11]. This precise system allows P. putida to transition from lag phase to exponential phase in response to the presence of V. dahliae. So, without the presence of the target fungus, the bacterial population wouldn’t be able to grow and spread. For more information, you can visit our Project Design page.

Figure 2: A gate activated by the presence of Ave1, which is a V. dahliae’s protein effector. In absence of Ave1/ absence of V. dahliae (left): Ve1 receptor is inactivated,  the OmpR protein does not have a phosphate group, ompC promoter is inactivated and DnaA is not produced. That leads to low rates of bacteria proliferation. In presence of Ave1/ presence of V. dahliae (right): Ve1 receptor is activated,  the OmpR protein has a phosphate group, ompC promoter is activated and DnaA is produced. That leads to high rates of bacteria proliferation.

The second biocontainment approach  is double auxotrophy. Auxotrophy refers to the inability of a microorganism to synthesize one or more essential growth factors, and will not grow if it’s not externally supplied [12,13]. This method is widely used as a biocontainment strategy. We chose vitamin B12 and L-tyrosine auxotroph.  Firstly, Vitamin B12 (cobalamin) is an essential cofactor required by over a dozen enzymes in bacteria and can only be synthesized by a limited number of microorganisms, including P. putida, as shown in Figure 3 [14]. With mutation in cobS (coding the Cobalamin synthase) and pduO (coding the ATP:cob(I)alamin adenosyltransferase)  we aim to create a strain that relies on external application. 

Figure 3: Biosynthesis pathway of Vitamin B12. By deleting the cobS and pduO we achieve a Vitamin B12 auxotroph.

Moreover, L-tyrosine is an amino acid required for protein synthesis and metabolic processes, but it is not abundant in most soil environments, ensuring that P. putida cannot thrive without supplementation [15]. With mutations in the tyrA  (coding the TyrA enzyme) and phhA  (coding the Phenylalanine-4-hydroxylase) genes we block the direct tyrosine biosynthesis pathway and the indirect through L-phenylalanine, as depicted in Figure 4, creating an L-tyrosine auxotroph [16]. 

Figure 4: Biosynthesis pathway of L- Tyrosine from Chorismate. By deleting the tyrA and phhA we achieve an L-tyrosine auxotroph.

This double auxotrophy is a highly effective biocontainment strategy because it makes it much more difficult for evolutionary processes to bypass containment. In this approach, the genetically modified organism is engineered to be dependent on two distinct, essential nutrients that it cannot synthesize on its own. For the organism to survive without external supplementation, it would need to acquire mutations that restore both biosynthetic pathways—a highly unlikely event compared to overcoming a single auxotrophy. This dual dependency ensures tighter control, as the absence of either nutrient prevents survival, significantly reducing the risk of environmental escape or unintended proliferation.  Additionally, tyrosine and Vitamin B12 can be supplied through our irrigation system, ensuring precise control over the survival of our engineering bacteria. By dissolving the required nutrients in the irrigation water, the bacteria are activated only when and where the solution is applied. This means that the bacteria's activity is not only confined to specific locations but also limited in time due to when the nutrients are depleted, the bacteria become inactive, ensuring they cannot persist or spread beyond the intended period of application. This temporal control adds a layer of containment, as the engineered bacteria will only function for a defined window, minimizing long-term environmental impact. For more information head over to our Implementation page.

~References~

[1] https://www.emro.who.int/health-topics/biosafety/index.html

[2] Zobel, S., Benedetti, I., Eisenbach, L., de Lorenzo, V., Wierckx, N., & Blank, L. M. (2015). Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida. ACS synthetic biology, 4(12), 1341–1351. https://doi.org/10.1021/acssynbio.5b00058

[3] Huber, N., Andraud, M., Sassu, E. L., Prigge, C., Zoche-Golob, V., Käsbohrer, A., D'Angelantonio, D., Viltrop, A., Żmudzki, J., Jones, H., Smith, R.P., Tobias, T., Burow, E. (2022). What is a biosecurity measure? A definition proposal for animal production and linked processing operations. One Health (Amsterdam, Netherlands), 15(100433), 100433. https://doi.org/10.1016/j.onehlt.2022.100433

[4] https://www.phe.gov/s3/BioriskManagement/biocontainment/Pages/default.aspx#

[5] Molina, L., Segura, A., Duque, E., & Ramos, J. L. (2020). The versatility of Pseudomonas putida in the rhizosphere environment. Advances in applied microbiology, 110, 149–180. https://doi.org/10.1016/bs.aambs.2019.12.002

[6] Halvorsen, T. M., Ricci, D. P., Park, D. M., Jiao, Y., & Yung, M. C. (2022). Comparison of kill switch toxins in plant-beneficial Pseudomonas fluorescens reveals drivers of lethality, stability, and escape. ACS Synthetic Biology, 11(11), 3785–3796. https://doi.org/10.1021/acssynbio.2c00386

[7] Rottinghaus, A. G., Ferreiro, A., Fishbein, S. R. S., Dantas, G., & Moon, T. S. (2022). Genetically stable CRISPR-based kill switches for engineered microbes. Nature Communications, 13(1), 672. https://doi.org/10.1038/s41467-022-28163-5

[8] Stirling, F., Bitzan, L., O’Keefe, S., Redfield, E., Oliver, J. W. K., Way, J., & Silver, P. A. (2017). Rational design of evolutionarily stable microbial kill switches. Molecular Cell, 68(4), 686-697.e3. https://doi.org/10.1016/j.molcel.2017.10.033

[9] Song, Y., Liu, L., Wang, Y., Valkenburg, D. J., Zhang, X., Zhu, L., & Thomma, B. P. H. J. (2018). Transfer of tomato immune receptor Ve1 confers Ave1-dependent Verticillium resistance in tobacco and cotton. Plant biotechnology journal, 16(2), 638–648. https://doi.org/10.1111/pbi.12804

[10] Kenney, L. J., & Anand, G. S. (2020). EnvZ/OmpR Two-Component Signaling: An Archetype System That Can Function Noncanonically. EcoSal Plus, 9(1), 10.1128/ecosalplus.ESP-0001-2019. https://doi.org/10.1128/ecosalplus.ESP-0001-2019

[11] Menikpurage, I. P., Woo, K., & Mera, P. E. (2021). Transcriptional Activity of the Bacterial Replication Initiator DnaA. Frontiers in microbiology, 12, 662317. https://doi.org/10.3389/fmicb.2021.662317

[12] Walker, G. M. (1999). FERMENTATION (INDUSTRIAL) | Media for Industrial Fermentations. In Encyclopedia of Food Microbiology (pp. 674–683). https://doi.org/10.1006/rwfm.1999.0575

[13] Arboleda-García, A., Alarcon-Ruiz, I., Boada-Acosta, L., Boada, Y., Vignoni, A., & Jantus-Lewintre, E. (2023). Advancements in synthetic biology-based bacterial cancer therapy: A modular design approach. Critical Reviews in Oncology/Hematology, 190(104088), 104088. https://doi.org/10.1016/j.critrevonc.2023.104088

[14] Hedo Berrocal, R., Martínez Sánchez, I., & Nogales Enrique, J. (2021). Engineering Pseudomonas putida for increased vitamin B12 production. Biosaia: Revista De Los másteres De Biotecnología Sanitaria Y Biotecnología Ambiental, Industrial Y Alimentaria, (10). Recuperado a partir de https://www.upo.es/revistas/index.php/biosaia/article/view/5806

[15] Schwander, L., Ligterink, N. F. W., Kipfer, K. A., Lukmanov, R. A., Grimaudo, V., Tulej, M., de Koning, C. P., Keresztes Schmidt, P., Gruchola, S., Boeren, N. J., Ehrenfreund, P., Wurz, P., & Riedo, A. (2022). Correlation Network Analysis for Amino Acid Identification in Soil Samples With the ORIGIN Space-Prototype Instrument. In Frontiers in Astronomy and Space Sciences (Vol. 9). Frontiers Media SA. https://doi.org/10.3389/fspas.2022.909193

[16] Molina-Henares, M. A., García-Salamanca, A., Molina-Henares, A. J., de la Torre, J., Herrera, M. C., Ramos, J. L., & Duque, E. (2009). Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440. Microbial Biotechnology, 2(1), 91–100. https://doi.org/10.1111/j.1751-7915.2008.00062.x