A major challenge in synthetic biology is the containment of genetically modified organisms (GMOs) outside of the laboratory. This includes restraining the physical spread of the organism and transfer of synthetic genes, via horizontal gene transfer (HGT). This challenge remains as regulatory bodies require sound evidence on the safety of GMOs which is difficult due to the lack of knowledge upon release. Additionally, the absence of risks is not necessarily a definitive proof of safety. Therefore, industry is unwilling to invest and scientists only rarely get permission to gather large-scale data. Here, DOPL LOCK can make the difference: we propose a mutually dependent double plasmid lock which will minimize HGT and the spread of GMOs. Our goal is to provide an open-source, standardised, modular and widely applicable Safe-by-Design biocontainment system for GMOs in semi-contained applications. With DOPL LOCK, we aim to drive bio-safety innovations, accelerating the field of synthetic biology.


Our world is being ravaged by global issues that manifest in local disasters, such as water pollution, climate change, and microplastic accumulation in nature. Although awareness among the public, policy makers, and the private sector is increasing, we as humanity are far from solving these issues. Every year, hundreds of iGEM teams come up with innovative ideas using synthetic biology to solve these issues and create a better future. Academia and the industry also acknowledge the potential of the field with great investments [1-4]. However, most of these groundbreaking solutions will never see the light of day since the required release of genetically modified organisms (GMOs) into the environment is strictly regulated in most parts of the world due to biosafety concerns. To meet the requirements for a license, strict guidelines are put in place by regulators. The extra layer of engineering to design a biosafety solution is complex and costly, making the implementation of synthetic biology in the real world a challenging task. Furthermore, while the building blocks for a robust biocontainment and biosafety solution may be present like auxotrophic markers [5] and the Deadman killswitch [6], engineering these building blocks into a well-thought-out system requires expertise, creativity, and inventivity. This has inspired us to develop a standardized, modular and implementable system to prevent horizontal gene transfer and ensure biocontainment. Our system could be used by startups, scientists, large biotech companies and iGEM teams to achieve the full potential of their innovations.

Horizontal gene transfer and biocontainment

Engineering bacteria may have massive potential, but the risks and concerns for the environment are significant as well. The main risks are (i) escape of GMOs into the wild where they could outcompete native species [7,8] and (ii) horizontal gene transfer of synthetic genes to naturally occurring species [5]. Both risks could bring the natural ecosystem in disbalance, potentially creating a disaster in the ecological cycle [8]. For example, a GMO could escape a bioreactor, spread through the environment and outcompete native species. Horizontal gene transfer is a common phenomenon among microbes to transfer DNA by transduction (transfer of DNA through bacteriophages), conjugation (exchange of DNA through the use of specialized proteins) and transformation (uptake of DNA from the environment by bacteria) [9,10].

The first method of horizontal gene transfer is through transduction, which occurs through bacteriophages [9,11,12]. After infection by a specific phage, genetic material of the bacterium may integrate into the phage's genome. This could lead to horizontal gene transfer of this integrated material to a bacteriophage-susceptible bacterium [9,11,12,13]. After infection, new phage particles will be produced in the bacterium, which at a certain point will kill the bacterium. These new phage particles are released into the environment and will infect other bacteria, creating a fast spread of the genetic material [9,12]. Infected bacteria are likely to be killed because of infection, which might limit the risks of horizontal gene transfer through this mechanism.

The second method is conjugation. Bacteria share their DNA through specialized proteins [9]. This type of DNA exchange is associated with plasmids, most likely because the DNA needs to be conjugated quickly as the transfer of the entire genetic element is needed for it to be functional. Conjugation of an entire chromosome would - depending on various factors including chromosome size and bacterial species - take around one hour, which is unlikely to complete [9]. Removing the Origin of Transfer (OriT) of a plasmid might reduce the chance of conjugation [14].

Finally, DNA could be released into the environment through transformation. A bacterium might lyse and release a plasmid or eliminate plasmids on its own into the environment, which could be picked up by other bacteria [9]. Preventing the transformation of free DNA from the environment has proven to be particularly challenging, since extracellular DNA can be detected in the environment even months after cell death, depending on the conditions [15].

For naturally occurring bacteria, horizontal gene transfer might not be a big concern, but this phenomenon quickly becomes a significant problem when engineering GMOs. Engineered microbes could have various advantages, such as a metabolic advantage over other native species. This means that these GMOs might be able to dominate existing species and bring the natural ecosystem in disbalance. Even if these GMOs do not escape their site of action, they could horizontally transfer their synthetic genes. This is the case in various hospitals, in which horizontal gene transfer of antibiotic resistance genes has been observed [16]. As a result, hospital-acquired infections are becoming more and more challenging to be treated with traditional antibiotics [17]. Similar developments could also occur in the environment if the release of GMOs for out-of-lab applications becomes a reality.

To prevent these ecological disasters, strict regulations and standards are in place for permits and licenses for semi-contained applications of GMOs [18]. Consequently, biosafety solutions are required to ensure GMOs do not escape into the wild or exchange their synthetic genes. These strict requirements form a layer of challenging engineering on top of the engineering process of the proposed solution and application. Designing an entire biosafety solution besides developing an environmental application of GMOs is costly and forms a massive barrier for implementing ingenious solutions. With our project, we aimed to develop a system that is modular and standardized to help scientists with the engineering of the biosafety part of their solutions, creating a Safe-by-Design system that prevents horizontal gene transfer and GMO escape. With such a system as the foundation, anyone from startups to industry-leading teams, can build reliable products that can stand the test of time.

Responsibility gap

The above-mentioned safety considerations are not the only reason synthetic biology applications have not yet been implemented outside of the lab. So we asked ourselves the question: With the potential of synthetic biology to solve current local, regional and global issues, how come these obstacles are not yet tackled? During our project, we identified a vicious circle which is caused by the responsibility gap [19].

In the development and implementation of synthetic biology applications we can identify three main stakeholders. These stakeholders have their own roles in the responsibility gap, with their own concerns, needs, and interests.

The first stakeholders are scientists. They are in charge of creating knowledge, developing new approaches and assessing containment and biosafety solutions. Nevertheless, the research of scientists is often limited to what is possible within the laboratory, and almost never on the scale of industrial applications. We have discussed this limitation with various experts during our interviews in our Integrated Human Practices page. Some scientists believe their methods could be used as proof of safety, while others suggest the laboratory environment is too simplified to draw conclusions on the efficacy of biosafety solutions. Scientists can test biosafety solutions in the laboratory but the specificity in the lab is limited, which makes extrapolating results from the laboratory to out-of-lab applications difficult.

The second stakeholder is the industry. Many biotechnology companies and corporations where synthetic biology could be applied, such as wastewater treatments, consider the costs to develop implementable synthetic biology solutions too high. In addition, they indicate that regulators are rarely satisfied with the biosafety measures they propose. Thus, the industry and many scientists ask for more lenient regulations to open these doors.

The last major stakeholders are regulators. They argue that safety is their number one priority. Understandably, experiments and data are needed to assure biosafety and this data should come from the industry or scientists. As long as the data is not present, it would be irresponsible to approve the semi-contained utilization of GMOs.

The responsibility gap arises when none of the stakeholders feel responsible to develop a biosafety solution that is implementable in the real world and test it on a large scale. To summarize, scientists develop and test systems in the laboratory, with all of the laboratory limitations, while the industry asks regulators for licenses to test and implement synthetic biology applications. The regulators cannot approve license applications due to uncertainty on biosafety. The developed biosafety solutions are currently not tested at a big industrial scale. This vicious circle has withheld the world from synthetic biology and its impact. To break this deadlock, the 2021 Leiden iGEM team has developed a new solution that could address the concerns of scientists, industry, and regulators. Our solution not only brings an innovative and elegant system to the field, but also describes how to build bridges between the three stakeholders, overcoming the responsibility gap. Our Double Plasmid LOCK, DOPL LOCK, is a Safe-by-Design, standardized, and modular foundation which would enable scientists, startups, and the industry to make the world a better place.


To make promising synthetic biology applications a reality, we aimed to develop a biosafety solution that tackles horizontal gene transfer and ensures biocontainment. But just a system is not sufficient and impactful enough. To form a bridge between scientists, industry, and regulators, we envisioned a system that is instrumental for change and meets the following criteria:

  1. Standardized: through standardization, the system is easy to use in combination with various other components. In addition, standardization of a biosafety solution provides a Safe-by-Design foundation for others to build upon;
  2. Modular: by making our system modular, we can support a diverse range of applications. All synthetic biology applications are different and have specific needs. Modularity gives engineers the tools to tweak the design while keeping a robust Safe-by-Design foundation;
  3. Implementable: a complex biosafety solution might be highly effective on paper, but if it is not implementable, its impact will be limited. By keeping regulations in mind, considering the commercial viability of such a system and ensuring the effectiveness of our DOPL LOCK system, we can work towards a foundation that can actually be implemented.

With these goals and requirements in mind, we have developed an interdependent double plasmid system using toxin-antitoxin systems and inducible promoters to prevent horizontal gene transfer and to ensure biocontainment. In our DOPL LOCK system, pRomeo contains toxin A and antitoxin B, while pJuliet contains antitoxin A and toxin B. The antitoxin and corresponding toxin are placed on separate plasmids, creating a dependence between the two plasmids. It is the classic Romeo and Juliet love story - our two plasmids just cannot live without each other! This is the first lock in our DOPL LOCK system. Additionally, the double plasmids are locked to a specific environment through inducible promoters. The antitoxins on each plasmid are transcribed using inducible promoters. If the inducer is present, the antitoxin will be transcribed and function accordingly. If the GMO escapes the designated site, the inducer is no longer present. Consequently, the antitoxin will not be transcribed and the GMO dies from the expressed toxins.

Toxin-antitoxin systems are found to be abundant in various bacterial plasmids and genomes [20]. The toxin causes inhibition of the growth of the bacteria, while the antitoxin neutralizes the toxin in normal conditions. In natural toxin-antitoxin systems, the antitoxin is degraded when signals of stress are present, leading to the stable toxin expression and inducing a state of dormancy [21].

Six classes of toxin-antitoxin systems are known, which all have different modes of action [21].

  1. Class 1 inhibits the toxin translation through binding to the mRNA of the toxin. The antitoxin sRNA binds to the mRNA of the toxin, preventing translation of the toxin.
  2. In Class 2, both toxin and antitoxin are expressed and translated, but in normal conditions bound to each other until the antitoxin is cleaved off due to stress signals.
  3. Class 3 involves antitoxin bacterial small RNAs (sRNA). In contrast to class 1, the toxin RNase interacts with the sRNA to form RNA pseudoknot–toxin complexes. This inhibits the toxin activity.
  4. Class 4 relies on fully translated proteins. In normal circumstances, the antitoxin stabilizes bacterial filaments, while the toxin destabilizes the filaments under stress signals, inhibiting cellular division.
  5. With Class 5 toxin-antitoxin systems, the antitoxin GhoS RNase targets the GhoT mRNA of the toxin. During stress signals, the antitoxin mRNA is degraded resulting in membrane lysis by the toxin.
  6. Class 6 involves the antitoxin protein marking the toxin protein for degradation. If the toxin is not degraded, DNA transcription is inhibited by the toxin.

pSEVA plasmid design of DOPL LOCL

Figure 1: The proposed pSEVA architechture of a DOPL LOCK plasmid. Two of these plasmids are needed for the DOPL LOCK biosafety solution.


In order to build a Safe-by-Design foundation for synthetic biology applications, we propose a final design that adopts the Standardized European Vector Architecture (SEVA) plasmid nomenclature. Adopting their standards means our system and proposed plasmid designs could more easily be used by researchers, startups, and the industry. The Origin of Transfer (OriT) of the SEVA plasmid is replaced by our safety module consisting of toxin A and antitoxin B with the appropriate promoters. More information is given on our Proof-of-Concept and Entrepreneurship pages.


Each synthetic biology application could be different and the freedom to adapt a biosafety solution is pivotal to eliminate case-specific risks and meet case-specific needs. The toxin-antitoxin combinations could, for example, be changed if required. During our lab time, we tested four combinations. In addition, the inducible promoter of the antitoxins can be changed as well. For example, in certain applications it might not be possible to use chemically inducible promoters, for which light inducible promoters with specific wave-lengths might be an alternative (primarily wavelengths outside of the spectrum of sunlight).


Although various interesting biosafety components exist, a complete system should be as simple as possible. During our Human Practices interviews, we learned that more complex systems are not necessarily better. In addition, we chose to use plasmids instead of relying on genomic integration, after careful considerations through experimental design and Human Practices. Furthermore, we analyzed the European regulation on out-of-lab use of GMOs and designed our system considering those regulations. On the Implementation page, we describe how our system addresses these regulations with two case-studies.

Finally, our project goes beyond designing a system. To break the deadlock of the responsibility gap, a bridge should be built between scientists, the industry, and regulators. Our Entrepreneurship plan describes how we can be a change agent with our DOPL LOCK system being instrumental in forming a bridge between different stakeholders, filling the responsibility gap, and revolutionizing synthetic biology by making it reality.

Below, you can see the proposed plasmid design with a description of each component if you click on it.

Origin of replication (Ori)

The Oris of the double plasmids should be compatible with each other and not compete for the same replication mechanism. According to our own results, the Oris p15A and pBR322/ROB are maintained at a similar level and thus a good option for our system.


The Oris of the double plasmids should be compatible with each other and not compete for the same replication mechanism. According to our own results, the Oris p15A and pBR322/ROB are maintained at a similar level and thus a good option for our system.

Promoter for the toxin

The toxins will be placed under the control of constitutive promoters, which ensures that the toxins are always expressed. If the GMO transfers to a region where the antitoxin is not induced then the constitutively active toxin will kill the organism, thereby preventing the spread of the GMO.

Promoter for the antitoxin

The antitoxins will be placed under the control of inducible promoters since that will allow us to predefine the conditions for GMO survival. To induce the antitoxin in many different environments and applications, there are multiple options to choose from: the promoters can be chemically-inducible, light-inducible, and pH-inducible [3,4,5]. Chemically-inducible promoters are easy to work with, but we have concluded some possible downsides, depending on the environment. Specifically, we have identified three main downsides: (i) it is not desirable in some environments to add chemicals to it, (ii) in some environments there may be naturally occurring chemicals that can interfere with the inducer, and (iii) the inducer could be degraded by naturally occurring enzymes in the environment. The pH- and light-inducible promoters can provide a more stable induction in these cases.


The choice for which TA system to use depends on both the environment the GMO will be released in as well as the host organism. The antitoxin will be used to ensure the GMO cannot leave the predefined area. Therefore, it should be metabolised quickly to ensure the GMO will be poisoned fairly quickly after the induction of the antitoxin stops.

Selection cassette

Even though antibiotic resistance genes are an easy selection cassette, it is not ideal with the application of DOPL LOCK in a semi-contained manner due to the global issue of multi-drug resistant bacteria. Therefore, this cassette will be switched for a split GFP on both of the SEVA plasmids [6]. When both plasmids are present in the cell, the GFP parts can reattach and the cell will be fluorescent green, which allows for selection of transformed bacteria.


Here the required genes for the functionality of the GMO can be placed.

Conclusion and future prospects

To break the deadlock and unlock the potential of synthetic biology, our DOPL LOCK project needs to be more than just a system. Not only have we created a foundation that could be used by others, we have also developed implementation and entrepreneurship plans to act as a change agent. By bringing the field of synthetic biology in movement through standardization, bridge building, and focusing on a Safe-by-Design approach, we tried to contribute to realizing synthetic biology applications. Synthetic biology has a great potential and we envision synthetic biology actually starting to impact our lives and solve local, regional, and global problems.

Our iGEM project is an important first step but further research, investments, and work are needed to fully realize the potential of synthetic biology. Due to the COVID-19 pandemic, our lab time and resources were limited. During our lab time, we were able to show the potential of the toxin-antitoxin system and the best combination of origins of replications for the double plasmids. More research is needed on testing the DOPL LOCK system for horizontal gene transfer, assessing the biocontainment rate and applicability in other organisms. More research and experiments are proposed in our Proof-of-Concept page. With our iGEM project as the first step, we aim to build towards a world in which iGEM teams, startups, and the industry can solve real problems, with real science, and real applications.


iGEM Leiden is not claiming that DOPL LOCK should be used for the release of genetically engineered organisms and therefore is not liable for any damages arising from the unsafe release of GMOs.

With DOPL LOCK we want to show that it might be possible to utilize GMOs outside of the lab in a safe and responsible manner. You should always consult a professional adviser beforehand and contact the government to get insight in GMO regulations. Before using GMOs outside of a biosafety environment.

During our project all the experiments were performed in a laboratory environment and no GMOs were released in the environment.


  1. DeLisi, C., Patrinos, A., MacCracken, M., Drell, D., Annas, G., Arkin, A., Church, G., Cook-Deegan, R., Jacoby, H., Lidstrom, M., Melillo, J., Milo, R., Paustian, K., Reilly, J., Roberts, R. J., Segrè, D., Solomon, S., Woolf, D., Wullschleger, S. D., & Yang, X. (2020). The Role of Synthetic Biology in Atmospheric Greenhouse Gas Reduction: Prospects and Challenges. BioDesign Research, 2020, 1–8.
  2. Howard, T. P., Middelhaufe, S., Moore, K., Edner, C., Kolak, D. M., Taylor, G. N., Parker, D. A., Lee, R., Smirnoff, N., Aves, S. J., & Love, J. (2013). Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. Proceedings of the National Academy of Sciences, 110(19), 7636–7641.
  3. Massachusetts Institute of Technology. (2019, 11 december). Synthetic biology: A new tool to tackle climate change? MIT Global Change [Persbericht].
  4. Cumbers, J. (2021, 2 februari). New Synthetic Biology Venture Fund Puts 'Scientist Founders' First. Forbes.
  5. Wright, O., Delmans, M., Stan, G. B., & Ellis, T. (2014). GeneGuard: A Modular Plasmid System Designed for Biosafety. ACS Synthetic Biology, 4(3), 307–316.
  6. Chan, C. T. Y., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2015). "Deadman" and "Passcode" microbial kill switches for bacterial containment. Nature Chemical Biology, 12(2), 82–86.
  7. WILLIAMSON, M. (1992). Environmental risks from the release of genetically modified organisms (GMOs)–the need for molecular ecology. Molecular Ecology, 1(1), 3–8.
  8. International Union for Conservation of Nature. (2004, augustus). Genetically Modified Organisms and Biosafety: A background paper for decision-makers and others to assist in consideration of GMO issues1.
  9. Thomas, C. M., & Nielsen, K. M. (2005). Mechanisms of, and Barriers to, Horizontal Gene Transfer between Bacteria. Nature Reviews Microbiology, 3(9), 711–721.
  10. Wright, O., Stan, G. B., & Ellis, T. (2013). Building-in biosafety for synthetic biology. Microbiology, 159(Pt_7), 1221–1235.
  11. Soucy, S. M., Huang, J., & Gogarten, J. P. (2015). Horizontal gene transfer: building the web of life. Nature Reviews Genetics, 16(8), 472–482.
  12. Redondo-Salvo, S., Fernández-López, R., Ruiz, R., Vielva, L., De Toro, M., Rocha, E. P. C., Garcillán-Barcia, M. P., & De la Cruz, F. (2020). Pathways for horizontal gene transfer in bacteria revealed by a global map of their plasmids. Nature Communications, 11(1).
  13. Villa, T. G., Feijoo-Siota, L., Rama, J. R., Sánchez-Pérez, A., & Viñas, M. (2019). Horizontal Gene Transfer Between Bacteriophages and Bacteria: Antibiotic Resistances and Toxin Production. Horizontal Gene Transfer, 97–142.
  14. Haase, J., Lurz, R., Grahn, A. M., Bamford, D. H., & Lanka, E. (1995). Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex. Journal of Bacteriology, 177(16), 4779–4791.
  15. Aminov, R. I. (2011). Horizontal Gene Exchange in Environmental Microbiota. Frontiers in Microbiology, 2.
  16. Lerminiaux, N. A., & Cameron, A. D. (2019). Horizontal transfer of antibiotic resistance genes in clinical environments. Canadian Journal of Microbiology, 65(1), 34–44.
  17. Grundmann, H., Aires-de-Sousa, M., Boyce, J., & Tiemersma, E. (2006). Emergence and resurgence of meticillin-resistant Staphylococcus aureus as a public-health threat. The Lancet, 368(9538), 874–885.
  18. European Parliament. (2001, 17 april). Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. Eur-Lex.Europa.Eu.
  19. Asin-Garcia, E., Kallergi, A., Landeweerd, L., & Martins Dos Santos, V. A. (2020). Genetic Safeguards for Safety-by-design: So Close Yet So Far. Trends in Biotechnology, 38(12), 1308–1312.
  20. Yamaguchi, Y., Park, J. H., & Inouye, M. (2011). Toxin-Antitoxin Systems in Bacteria and Archaea. Annual Review of Genetics, 45(1), 61–79.
  21. Page, R., & Peti, W. (2016). Toxin-antitoxin systems in bacterial growth arrest and persistence. Nature Chemical Biology, 12(4), 208–214.">