Implementation

This section aims to contextualize the circuit and framework for their application in multiple areas and conditions, in addition to expanding on what that would entail.

Genetic engineering and synthetic biology are in constant development to improve society in multiple areas. One important fact that limits the implementation of many projects and ideas is the release of genetically engineered organisms, since it is a controversial topic that implies several concerns. Many countries have regulations regarding the release of engineered organisms and are developing strategies to ensure their safety towards the environment and the population. Some of these strategies are novel genetic manipulation techniques, complex biocontainment systems and new regulatory frameworks to start implementing engineered organisms in the environment. Engineered organisms have the potential to improve humanity to a new level, but modern methodologies, characterizations, risk evaluations, and staged proofs of concept are still necessary to completely apply engineered organisms and obtain their maximum capabilities hand to hand with the security of the environment, living organisms and society.

Reference: Bruetschy, C. (2019). The EU regulatory framework on genetically modified organisms (GMOs). Transgenic Research, 28(S2), 169–174.

Our project began with the idea to expand the use of engineering organisms based on targeting their release. Engineered organisms are applicable to any area of development and could help improve society in multiple fields, but their use in uncontrolled environments is restricted because of concerns regarding the release of engineered organisms. Their impact could include alteration of the environment, leak of genetic material into wild species, colonization of niches, diversity decrease and others. We want to change this perspective.

The circuit’s design was focused on its application, taking into account punctual requirements depending on the user’s specifications. If interested in the specific design objectives of our circuit itself, check out Description. Through our implementation proposal we aim to contextualize and create real strategies for the introduction of our genetic suicide circuit across the scientific borders, in addition to assess their possible and probable implications.

Finally, we highlight the advantages and functionality of a system like ours, which builds upon previous techniques and methodologies to keep expanding the barriers of science.

Our suicide circuit is focused on expanding the capacities of GMO´s by generating an efficient system to control an organism’s life, automatizing its death to a specific, previously determined moment. Suicide circuits, also known by the name of “kill-switches”, are biocontainment systems based on the death of an organism through a specific signal. Different types of kill-switches exist: activated by physical factors, induced by chemical molecules and some even activated by RNA interactions (iGEM Ohio State, 2020). Our system differs from the others in important aspects. First of all, our counter circuit enables the autonomous time management of our engineered bacteria, making the kill-switch independent from environmental conditions, contrary to most of the existing kill switches (Chan, C et al. 2016; Stirling, F et al. 2017). The counter is also based on sequential logic, quality which allows the system to generate different outputs from the same input, unlike combinatorial logic, in which the same input generates the same output. Additionally, the whole system is modular because each module can be applied and modified individually depending on the purpose, which grants it robustness, and plenty of personalization and optimization possibilities. If you want to further understand our circuit’s design please visit our {description page}.

References:
-iGEM Ohio State (2020). Biocontainment Reimagined. iGEM Team Ohio State. https://2020.igem.org/Team:OhioState
-Chan, C. T., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2016). 'Deadman'and'Passcode'microbial kill switches for bacterial containment. Nature chemical biology, 12(2), 82-86.
-Stirling, F., Bitzan, L., O’Keefe, S., Redfield, E., Oliver, J. W., Way, J., & Silver, P. A. (2017). Rational design of evolutionarily stable microbial kill switches. Molecular cell, 68(4), 686-697.

The effectiveness of our genetic suicide circuit is directly related to its components, which we have thoroughly assesed. The counter circuit, core of our device proposal, is based on sequential logic, which makes it highly effective in comparison to other approaches as detailed in our comparison section. Our design also allows for very in-depth characterization and successful troubleshooting, as proposed in Description.

Our suicide circuit is oriented towards fulfilling the need for an autonomous limited life organism that performs a job, avoiding or diminishing the consequences caused by the remanence of engineered organisms in an uncontrolled enviroment. This becomes a way to prevent environmental harm while allowing the tackling of complex problems through one of the best tools humanity possesses: synthetic biology.

A counter is a tool that can be used in multiple areas to count a specific event or situation and obtain information about the count for certain purposes depending on its use. Counters based on biological elements have been developed across the years and have many applications in the scientific community, but the capabilities of a biological counter haven't been expanded and implemented completely. (Subsoontorn & Endy, 2012). A fully functional biological counter could work to count events like the proliferation of different kinds of cells, to determine how many are resistant to antibiotics in a subpopulation or even count difficult events like circadian cycles. Our suicide circuit contains a counter module that tracks time through cell division, this mechanism allows to keep track of the different stages of a bacterial population and the triggering of a determined event after the final count has successfully been reached. In our design, this specific event is a controlled death mechanism; however, this could easily be replaced by any other functional module, such as triggering specific metabolic pathways. Applications for an organism with limited life are countless and with our suicide circuit this can start to be implemented, in the next section we described some applicatations for our system and its functionality. The counter’s characteristics are described in Description.

Reference: Subsoontorn, P., & Endy, D. (2012). Design and analysis of genetically encoded counters. Procedia Computer Science, 11, 43-54.

Our suicide circuit can be applied in many areas once it is further improved for a specific application. Many tasks need a limited life organism to make more efficient and overall improve a certain process or system; with our circuit these jobs could be carried out. We based ourselves on experts feedback, literature and iGEM projects proposal to choose four areas to explain and demonstrate the utility of our circuit that are described below.

Bioremediation

We all know that the contamination around the world is always increasing. Oil spills, heavy metals in the soil and pesticides are some of many contaminants that affect the environment according to the World Health Organization (WHO). (Philp, 2015) Genetically engineered organisms can improve bioremediation techniques but are limited because regulatory policies regarding their use in uncontrolled environments are not completely developed, and their environmental impact is still a cause for concern.

We could apply our system for specific bioremediation applications, such as wastewater treatment; particularly for the elimination of hazardous compounds or for bioremediating a contaminated environment in situ to avoid loss of balance and natural properties. Our system could also complement existing proposals, just as iGEM NAU-China 2020 project, that aimed for the remediation of heavy metals using earthworms and engineered bacteria. Its mechanism starts when the engineered bacteria leave the intestine of the earthworm and, in this environment, don't have a specific protein and RNA of the earthworm, inducing the killing circuit. This makes the killswitch dependent on its environment, which could backfire, since remnants of the specific protein in the bacteria may affect the efficiency of the circuit. Our circuit could automatize the death phase and make it independent from the environment.

We also talked with professionals that, themselves, suggested the application of our system for bioremediation aspects. M.Sc. Carola Scholz told us that our circuit has potential in bioaugmentation studies, water treatment techniques and environmental assays. PhD. Frank Solano Campos, Lic. Juan Carlos Hernández and Ph.D. Candidate Kattia Nuñez Montero also mentioned that the circuit could be applied in bioremediation, while M. Eng. Randall Chacón Cerdas saw an application for metals bioremediation specifically. Bioremediation was the most agreed upon application across our discussion with different experts and waster-water treatment was the most specific action of use mentioned by the specialists in this field.

Reference:
Pant, G., Garlapati, D., Agrawal, U., Prasuna, R. G., Mathimani, T., & Pugazhendhi, A. (2020). Biological approaches practised using genetically engineered microbes for a sustainable environment: A review. Journal of Hazardous Materials.
Kumar, S., Dagar, V. K., Khasa, Y. P., & Kuhad, R. C. (2013). Genetically modified microorganisms (GMOs) for bioremediation. In Biotechnology for environmental management and resource recovery (pp. 191-218). Springer, India.
Philp, R (2015.) BIOREMEDIATION: THE POLLUTION SOLUTION? Microbiology Society. https://microbiologysociety.org/blog/bioremediation-the-pollution-solution.html

Biomedicine and Therapeutics

Biomedicine and therapeutics are always improving due the new technologies based on biotechnology, genetic engineering and synthetic biology. These areas can expand the medical field but, according to Amrofell et al 2020, genetic tools need to be developed to implement engineered organisms as smart treatments and novel therapies to achieve the application of synthetic biology in medicine areas. Engineered organisms have been a focus of attention for biomedical applications, such as diagnosis systems and treatment tools for specific diseases, and could be great platforms to improve medical strategies (Ruder et al. 2011). Our circuit offers a new possibility to include methodologies based on synthetic biology in the medical field. Currently, the development of these applications is based on generalistic treatments, invasive methodologies and robust techniques of operation (Bober et al. 2018); we want to implement our circuit to facilitate these processes by providing a novel mechanism of cellular death into engineered organisms to target specific diseases and to develop new therapies based on the patients needs.

Our system enables a brand new machinery to implement these ideas to the real world due to their intrinsic modules that are not found in other killing or biocontainment systems, such as its autonomy, it's mechanism based on sequential logic and the evolutionary stability of the modules. We also expect that our circuit complements promising ideas and projects regarding this area like iGEM UNILausanne 2020 project. They aimed to make a treatment based on probiotics to target colorectal cancer, in this case our system could work as a death mechanism to finish the job of the bacteria in a very useful way. Experts such as M. Sc. Silvia Castro Piedra valued the impact of our tool in the area, and suggested that a system like ours could function in an interesting way to attack pathogenic infections, to prevent severe immune responses, to develop responsive macrophages that differentiate as needed, to create extracellular matrices without the need of synthetic materials, and even fibrosis and COVID-19 treatments. PhD. César Rodríguez Sánchez and Lic. Rossy Guillén Watson saw the applicability of the circuit for making in vivo assays regarding interaction between bacteria and the organism, and for experimentation with different pathogenic bacterias, respectively. PhD. Rodrigo Mora Rodríguez told us that it could be interesting to analyze the mechanism of action of new types of antibiotics, while Dr. rer. nat. Miguel Rojas suggested the use of this technology to develop effective in vivo models for drugs and other compounds of interest. The biomedical field is an area of expansion ready for the implementation of synthetic biology systems and we aim to do it with our circuit.

Reference:
-Amrofell, M. B., Rottinghaus, A. G., & Moon, T. S. (2020). Engineering microbial diagnostics and therapeutics with smart control. Current opinion in biotechnology, 66, 11-17
-Ruder, W. C., Lu, T., & Collins, J. J. (2011). Synthetic biology moving into the clinic. Science, 333(6047), 1248-1252.
-Bober, J. R., Beisel, C. L., & Nair, N. U. (2018). Synthetic Biology Approaches to Engineer Probiotics and Members of the Human Microbiota for Biomedical Applications. Annual Review of Biomedical Engineering, 20(1), 277–300.

Industry

The industrial area has been improved by synthetic biology across the years. Many areas in the industry, like agriculture development, biofuel synthesis, pest control systems and the production of diverse compounds like metabolites of interest and drugs got more efficient due to the use of engineered organisms. Methodologies based on the use of synthetic biology and engineered organisms for industry aspects have been a great tool to upgrade the industry itself, but, can we go further? We think that our circuit could allow new mechanisms for diverse industry processes either to improve existing methodologies to increase their efficiency and applicability or to establish new ones due to the intrinsic characteristics of our construct. Optimizing the conditions for the production of specific biomolecules, preventing their misfolding and aggregation; industrial processes involving intracellular metabolites and their timely liberation, along with the reduction of involved hardware and the independence from environmental conditions; and the production of determined food like beer or yogurt with desired conditions and properties, like the inability to isolate them from the final product, allowing a business model based on this exclusivity are some of the applications that our circuit could be used for in the industrial area. Other strategies could be developed from functions of interest such as the augmentation of bioavailability of phosphorus in soil, the ability to co-culture in fermentations in order to align resources, and the optimized trying out of different conditions. Also our system could complement industrial ideas like iGEM UM Macau 2020 project, they proposed an interesting system to remove the biofilm formation inside aquariums and ocean parks. Our circuit could complement their system in the moment when it’s necessary to remove the engineered bacteria after finishing its biofilm degradation job. Additionally, we received feedback from experts, PhD. Karla Meneses Montero and M. Sc. Raúl Trejos Espinoza, who suggested applications for our circuit in industry aspects. They mention that it could be very useful as a biocontrol system for bacteria and to diminish production costs in optimized systems as fermentations by improving strategies of use, respectively.

Biosensing

Biosensors are analytical tools that work to detect one or more specific compounds in a complex system such as an uncontrolled environment. Some organisms are engineered to be used in biosensing applications to target and detect molecules in a simpler and efficient way. Our circuit would allow the development of new systems of biosensing that are based on an expected death of the organism after the biosensing application. This is useful for applications such as detection of diseases that are difficult to prevent, toxic compounds’ detection in an open environment or to verify the quality of a molecule of interest. In some cases the biosensing of a sample can be difficult due to its complex calibration and detection limit, this can cause false negatives or false positives when we analyze a system with a biosensor; these factors have caused the implementation of biosensors to be slow. It is necessary to increase the robustness of these systems to incorporate its capacities and utilities across other fields.

Due to the killing mechanism of our circuit, all the parameters can be optimized to expect the death of the organism in a specific moment of the growth and development of the organism, in relation to its biosensing ability, as required. This can be applied and implemented to multiple projects that propose biosensors like the iGEM Hannover 2020 project. They aimed to design a biosensor that detects the formation of a biofilm in a patient’s implant to avoid the caused inflammations and infections across the body. We think that our autonomous suicide circuit could fit very well inside this project because it could allow the development of a limited life biosensor to incorpored it in a uncontrolled system like the human body. Dr.rer.nat Miguel Rojas Chaves suggested that a system like ours could work to further analyze how a specific bacteria reacts to certain components and to obtain effectiveness models of these compounds.

Reference: Turner, A. P. (2013). Biosensors: sense and sensibility. Chemical Society Reviews, 42(8), 3184-3196.

Risk Assessment

To identify potential risks and hazards, we made a risk assessment to analyze the potential affectations that our circuit could be involved in. It’s important to prevent and note the potential impacts that our technology could have, in order to establish its correct use and implementation. For the development of the risk assessment we followed several steps to perform it in the best way. First we identified the potential risks, in which we define circuits malfunction, environmental interactions, bioterrorism and the dual-use of our technology as our main risks. It is important to consider that no system is perfect, some issues can appear and affect the functionality of the circuit. External and internal factors could affect the mechanism of the circuit and it is crucial to denote these aspects and solve them to ensure the stability and robustness of the circuit. The environmental interactions of our engineered bacteria with the wild organisms in an uncontrolled system is also an extremely relevant issue to contemplate. Introducing an external organism into an open environment like a wetland or inside another organism causes the preoccupation of how the engineered organism can affect or influence other living beings in different forms. For bioterrorism, we came to the conclusion that society itself could get harmed due to the possibility of a bioweapon developed based on our circuit. For the dual-use of our technology we focused most on the applicability of the circuit. We discuss that our circuit could be useful to facilitate the improvement and innovation in areas of interest, but could also be misused due to its capacities and characteristics. Below we described in a more punctual way these four potential risks, their considerations and strategies to tackle them to ensure safety.

Circuits Malfunction

Biological systems can malfunction depending on the construct stability; the factors on their environment like pH, temperature or can affect the development and functionality of a system. If a factor is too strong it can disable the system completely. In our case, we need to ensure the robustness of the different modules of our system to archive its correct work. The initial repression, the count mechanism, the expand count and the kill-switch need to function as a whole to generate a correct use in a specific application. Internal factors like mutations and genetic material degradation can affect the circuit, this issue is assessed in {Description-Characteristics}. On the other hand, external factors could also affect the correct function of the system. It is necessary to consider that these elements are more unpredictable, and include the natural death of the engineered bacteria in the environment or the extrinsic affectations of the modules by the environmental parameters like pH. It is possible to reduce these affections by thoroughly analyzing the target environment in which the system is going to function and troubleshoot every possibility of malfunction in that specific area. The malfunction of the circuit is part of it and its development, but we can take actions to ensure its correct function and minimize the risk of failure of the system.

Environmental interactions

The introduction of engineered organisms into an open environment causes the interaction between the engineered organisms and the different parts of the ecosystem. Singh et al mention that efforts should be made to examine the performance of survival and potential gene transfer of engineered bacteria because these aspects can affect the endogenous organism in the environment. To tackle this type of issue it is important to include a robust biocontainment system to avoid leaks of genetic material into wild bacterias in the first place. Also it is crucial to determine all the overall risks that the engineered bacteria could have in the open environment and make multiple assays to see the interaction of the engineered organism with the parameters of the expected space of application. Characterizing the environment of implementation and making the correct optimization of the system makes it easier to anticipate potential interactions between the engineered organism and the environment. Taking into consideration all these factors would ensure the correct development and expected performance of the desired system. Singh et al also highlight that there's no evidence that engineered organisms have caused a significant adverse impact on the natural community but since the possibility exists and it is an important risk that we need to consider for the expected application and real implementation of our system.

Reference: Singh, J. S., Abhilash, P. C., Singh, H. B., Singh, R. P., & Singh, D. P. (2011). Genetically engineered bacteria: An emerging tool for environmental remediation and future research perspectives. Gene, 480(1-2), 1–9.

Bioterrorism

Bioterrorism is based on the use of biological elements to propagate death and damage to living beings. Biological elements like bacterias, viruses and toxins with specific characteristics are normally in the eye of bioterrorists to use them as hazardous weapons to affect the integrity of the society. We consider that our circuit could have a potential as a biological weapon, thus all the problems of bioterrorism should be contemplated as a safety aspect. In one of multiple worst scenarios our circuit could be implemented into a dangerous organism to spread a disease or toxin; then the organism would just be eliminated leaving no trace. The fight against bioterrorism is always in development; methodologies for regulations about dangerous pathogens, surveillance, detection, response and risk communication are in constant improvement to minimize bioterrorism and its consequences. We expect that our system will never be used as a biological weapon, but it is important to mark bioterrorism as a possible risk to ensure safety of our technology and society. To prevent these types of dangerous actions based on biological parts, it is important to develop a plan of action to reduce the risk of a bioterrorism attack. Also the divulgation of information generated by experts about these risks helps the understanding of the population to know how to act and minimize the issue.

Dual-use implementation

Dual-use implementation refers to the application of a technology for multiple objectives in a specific moment, where these objectives can be for good purposes or not. This aspect can promote the use of a system in a way not stipulated or expected, i.e. an incorrect use. This incorrect use or misuse can be generated by the potential capacities and intraspecific qualities of a technology. In our case, this issue could affect our circuit in practically any area. To make it more demonstrative, let's consider the bioremediation area. Having an organism incorporated with our circuit that degrades oil spills and then kills itself could be a great tool for treating this kind of contamination, but having this potential option to solve this kind of problem could allow the incorrect treatment of oil spills and even the loss of its importance if one occurs, just because there's a tool to solve it. This is just one example of many scenarios that can happen if the system is implemented in an incorrect way; it is possible to prevent this by establishing standardized protocols of use, promote information about the correct operation mode of these type of systems and explain that the special qualities of a technology are for help and improve society not for abuse of it. Communication, moral, awareness and follow-up of the system’s implementation are key for the prevention of this issue. We took it upon ourselves to analyze risks in an integral perspective, with the Next-Level Biosecurity: Dual Use Research of Concern Workshop organized by iGEM.

Reference: Marris, C., Jefferson, C., & Lentzos, F. (2014). Negotiating the dynamics of uncomfortable knowledge: The case of dual use and synthetic biology. BioSocieties, 9(4), 393-420.

Strengths

Autonomous system, our circuit doesn’t depend on determined external/environmental parameters like chemical or physical triggers
Limited life based system that allows the death control and lifespan of a specific organism
Use of sequential logic, we chose a different approach for the development of the genetic circuit that enables more efficiency
Modularity of the construct for case-based implementation
Applicable in multiple areas and ways

Weaknesses

Optimization and characterization is required to be specifically implemented for an application
It’s necessary to make multiple assays in order to determine the efficiency and reliability of the system
Possibility for DNA transfer into wild organisms in the environment and impact on the environment
RNA transcript length and synthetic DNA conformation
Promoter pulse might be insufficient
Natural death presents unforeseen consequences

Opportunities

The implementation of a new autonomous suicide circuit across the scientific community
Divulgation and explanation of sequential logic as a potential system that needs to be further explorer and can be applied in multiple applications
Promote the advantages of a system like ours to expand the benefits of synthetic biology across multiple areas
Due to the novelty of the system there's a lot of opportunities for its exploitation, optimization and implementation

Threats

Uncertainty of uncontrolled environments and their characteristics that can affect the functionality of the system
Regulations about GMO’s that can limit the potential of the system and delay the implementation of the circuit in real life applications
Potential users could prefer a more described and characterized suicide mechanism than ours
Unforeseen circumstances could bring about unforeseen consequences
Technology beginning to be explored
Reduced information about sequential logic systems and circuits like ours

How we are targeting those threats

Optimizating of the DNA sequence to prevent its misfolding
Beginning the characterization of the system to further explore the possibilities of using our circuit in an uncontrolled environment. Plan for its further characterization.
Reporting and communicating all the data of assays using our system, to study and prove the utility of the system and its advantages.
The system has a multi-biocontainment system for the genetic material. When the organism dies, all the DNA is degraded to avoid the exchange of genetic material between wild type organisms in the environment and our synthetic organism

Regulations

Regulations for the use and application of synthetic biology ideas is completely necessary. New tools and projects are in constant development, and their regulation is usually right behind, affecting their implementation and development. Regulations are established to ensure safety to the population and prevent any risk that can be caused by a synthetic biology system. However, due to the increase and expansion of science nowadays, regulations for more complex projects with great objectives are lagging behind due to ambiguity, bureaucracy and even the case-by-case studies required in most cases. For example the Cartagena Protocol on Biosafety, which was signed and applied by the international community in 2003 for the regulation of modified organisms across countries and allowed the expansion of use of modified organisms, but its incorporation to national legislation varies. Costa Rica’s being determined by political aspects as explained in {Human Practices-Shipping & Policy}. Additionally, for a specific project or idea it is necessary to evaluate the technology used for the development of the product because the regulations also depend on it. Besides that, each country decides the necessary studies and analysis that have to be done for proposing regulations, in which socioeconomic and sociopolitical aspects are considered depending on the focus. For the effective regulation and approval of novel technologies, developers need to communicate closely with regulators, get involved in their work and give as much support as possible, since without legislation, production and commercialization doesn't exist. It’s crucial to orient the legislation to establish new regulations as needed, but it’s also important to promote the process and development to keep improving in different areas hand in hand with regulations that can enable a real implementation of synthetic biology systems. The generation of new data is required to evaluate the products and optimize its regulation, and furthermore it’s necessary to fight for the accessibility of the technology, not only its development. For this and more aspects, the legislation needs to be constantly reviewed in order to continue expanding the implementation possibilities for great technologies.

References:
Guan, Z., Schmidt, M., Pei, L., Wei, W., & Ma, K. (2013). Biosafety Considerations of Synthetic Biology in the International Genetically Engineered Machine (iGEM) Competition. BioScience, 63(1), 25–34.
O´neal-Coto, K (Dicember 20, 2019). La región está por definir el tratamiento legal para los productos derivados de la edición de genomas. Universidad de Costa Rica. https://www.ucr.ac.cr/noticias/2019/12/20/la-region-esta-por-definir-el-tratamiento-legal-para-los-productos-derivados-de-la-edicion-de-genomas.html
Secretaría Nacional de Ciencia, Tecnología e Innovación de Panamá. (2021) Regulaciones internacionales sobre algunos productos biotecnológicos. Clayton, República de Panamá.
Green, M. S., LeDuc, J., Cohen, D., & Franz, D. R. (2019). Confronting the threat of bioterrorism: realities, challenges, and defensive strategies. The Lancet Infectious Diseases, 19(1), e2-e13.
Mariner, W. (2020). Bioterrorism Act: the wrong response. In Bioterrorism: The History of a Crisis in American Society (pp. 24-24). Routledge.

To analyze and define our potential end users we choose four main stakeholder areas that have a direct relationship with the proposed applications mentioned above. First we selected industrial stakeholders that incorporate food and agricultural producers, industry owners and multinational companies. Industry has great implementation potential due to their daily activities and functions of constant development. Next, we selected bioremediation stakeholders that include bioremediation companies and environmentally focused non-governmental organizations. Bioremediation methodologies are always in expansion to fight contamination, thus our suicide circuit has potential to be incorporated into new approaches of bioremediation techniques. Subsequently we chose biomedical stakeholders that include the pharmaceutical industry and private biomedical companies. The improvement of medical areas is expanding hand in hand with synthetic biology technologies due to this aspect, our circuit could be very useful for an implementation in biomedical aspects and its users. Finally, we pick governmental stakeholders: national biotechnology laboratories, state universities and national ministries are part of this section. Governmental users have the power to start applying our system in a real way, empower its development and could help us exceed limitations for the implementation of the circuit.

Stakeholder map

We made a stakeholder map as well, using the virtual tool “iGEMers Guide to the Future” (https://live.flatland.agency/12290417/rathenau-igem/) developed by a collaboration between iGEM teams and the European Mobilisation and Mutual Learning Action Platform SYNENERGENE. In this map we see our chosen stakeholders in a graph in which the x-axis means the interest of the stakeholder in our project and the y-axis means the power of the stakeholder to influence our project in a positive or negative way.

By making the stakeholder map and the analysis behind it, we obtain that a great option to start the implementation of our circuit would be in state universities. State universities could have a great interest in the system, we discussed the project with several experts from state universities and saw a relevant use for the circuit. This could also have a big impact and influence for our project due to its validation as approved by relevant entities, hence opening many doors for us. On the other hand, multinational companies and industry owners may perhaps not be as interested in the system due its complexity and, in a generalized way, they may not understand the utility of the circuit. Thus, if we expect to implement our circuit with these stakeholders, very effective communication must be prepared, along with the specification of benefits that they would obtain when working with this. Optimization of the system for specific applications would also be very valuable for these types of stakeholders. Policy makers who regulate the implementation of our proposal are another critical group with whom dialogue should be pursued when possible; the more involved the developer is, the less likely things are to go sideways. Other groups, such as environmental activists could even display a strong position against the release and use of engineered organisms, given the uncertainty of the consequences it has. Work should be done with both stakeholders in order to include them into the development process, optimizing the project and creating the right conditions for its carrying out.

Impact on scientific community

We mentioned above that our circuit could be implemented in different areas of research in a very useful way, but how could this have a real repercussion in the scientific community? With our circuit we aim to solve scientists’ problems, enabling them to keep improving society. As a tool, our construct can be used to start working on areas that have not been completely addressed so far, like complex environments. We also want to focus the perspective about the use and release of engineered organisms, they have great potential of use but due to concerns and regulations the applicability is limited. We expect to have an impact across the scientific community because with our circuit we can open many doors in this scope, explore new possibilities and approaches in a novel form that has not been done before.

Future approaches

Hand in hand with experts feedback, we also aim to implement our suicide circuit into new development areas and for advanced applications to keep expanting the applicabiliy of synthetic biology. First of all, the bioelectronics field, with our system, could be possible to generate or eliminate potential charges after the expected death of the organism, this for the controlled construction of biocircuits. Our circuit could be useful for the generation of synthetic extracellular matrices for the synthesis of specific metabolites and then expect cell death, to only adquiere the molecules of interest and eliminate any background interference compound. It could also be applicable to study events that are difficult to define like the determination of cytotoxicity, circadian cycles, planned obsolescence, oscillation frequencies, the quantification of bacterial proliferation and bacterial resistance to antibiotics. Plant varieties could also be developed and agriculture could be improved by adding bacteria that promote plant growth and don't generate metabolic issues.

Future snapshots

We made a future snapshots analysis to anticipate the utility and implementation of our circuit in a hypothetical future basing ourselves on the fact that our system was completely accepted into society, since we believe that although it may pose some concerns initially, slowly it will become accepted. First, we visualize an increase in different applications based on the use of engineering organisms with limited life. One of the conferences we attended even proposed that a whole industry would probably be built out of different organism liberation methods. We think that our system will be completely characterized and optimized as a tool that can be implemented to any idea or project. We consider that regulations about synthetic biology and the use of engineered organisms may be changed to establish aspects about the correct use of systems like ours. Finally, we want to be an inspiration to the scientific community and further projects that aim to keep improving synthetic biology and the society itself.

State machines are based on sequential logic, they respond to specific inputs to perform a certain output depending on the state of the system and the inputs history. In these types of systems, the architecture conformation of the machine is crucial for the response across different types of inputs; a same input can cause a completely different result depending on the state of the system. State machines are applicable to biological systems due to their flexibility, time response and memory, and can enable a whole new perspective of development for synthetic biology applications. Nevertheless, sequential logic is just beginning to be applied to biology, the construction of genetic circuits and the conceptualization of complex systems. For it to be implemented, the main requirements are a change of point of view and methodology of resolution. Visualizing cellular constructs and genetic circuits as state machines enables a different line of thinking to solve complex problems with novel, more efficient, solutions, yielding great opportunities of implementation in synthetic biology.

Reference: Madec, M., Rosati, E., & Lallement, C. (2021). Feasibility and reliability of sequential logic with gene regulatory networks. PloS one, 16(3).

When we started to explore sequential logic to apply it to the project, we encountered a lot of possibilities and decided to make available this perspective in order to expand the use of these types of systems. The development of our framework and software arised from the utility that sequential logic based systems can confer to synthetic biology, given the virtually unexplored opportunities granted by the similarities between biological systems and state machines. With our framework we aimed to facilitate and guide the performance of complex biological tasks; as well to implement it in a real way as a tool for modular simulation and analysis.

We designed our framework and software to make a tool that can guide, construct and assess diverse types of synthetic genetic circuits according to the user's wants and needs. Our inspiration was based on creating a general and modular system for the exploration of sequential logic based synthetic biology to benefit experts and scientists with different types of knowledge, in a novel way that can be applicable in many fields of action.

Sequential logic gives a new perspective for the resolution of complex scenarios and we want to take advantage of its characteristics to give the scientific community a novel tool based on this mindset. During our user interviews one of the interviewees even told us that she understood where we were going when we had only explained sequential logic in theory. With our framework the user can establish the conditions for different situations to occur in each specific state of action, this increases the generation of results and data of parameters of interest according to the application and visualizes their affectations on the overall model.

Frameworks, algorithms, software and databases for the construction and development of genetic circuits have been made across the years, but most of these projects and ideas are based on combinatorial logic. In a combinatorial logic system the output only depends on the present input; the state of the system is not relevant because the response is only mediated by the activation signal. This contrasts with sequential logic, where the state of the system affects the whole behavior and response of the system. We establish a conceptual framework based on sequential logic to construct and apply genetic circuits in the most efficient way according to the users wants and needs. This type of framework enables an efficient and modular approach to tackle sequentially dependent biological tasks. Additionally, developing a framework software allows the direct implementation of the conceptual framework, in which its characteristics such as its structure and behavior can be applied. The structure of the software gives the user a set of parts that can be incorporated into the system to provide specific organization of action to resolve the expected function; its behavior is validated through the interactions and functions of the parts with other biological elements inside the system to generate results and data of the expected genetic circuit. In addition, through its database we have the possibility to store the constructs from the software, making them easily available to be assessed by different kinds of users. To know more about our software, you can go to {best software tool}.

With our framework we expect to make an impact in the development of genetic circuits by giving examples, success scenarios and clear advantages of using a system based on sequential logic rather than other systems. We validated the software applicability via a two-step proof of concept: a modular and easily applicable framework software, and its implementation, the software tool. By proving its utility and effectiveness, we are getting closer to implement this type of approach into synthetic biology applications and increasingly improve science itself.

To better understand sequential logic and our framework, founded upon it, we want to first make an emphasis on how sequential logic can be applied and implemented to solve complex problems. Sequential logic allows us to visualize situations beyond input-output systems, due to the importance of the state. With sequential logic, the same input can cause a different output depending on the conformation of the system, like in biological processes. In biological systems, state is crucial for its function, interactions and development. Genetic expression and interaction with a substrate or even apoptosis are processes in which the same signal can induce a completely new behavior depending on the current state of the parts and the other elements that interact in the system. This is why sequential logic can be perfectly applied into biological circuits. By understanding this we can see a problem as a state machine, depending on the situation and specific moment. To solve it, optimizing and characterizing multiple scenarios of action between selected parts and the construct itself can be carried out. Having this mindset when we face a complex system or problems allows us to think in multiple ways to settle a solution if we see it as subsequent steps or decisions, giving sequential logic an authentic utility in the real world.

An open access or open source software is a type of modality in which a computacional software is released under a specific license. This license confers a more public application for the use and development of the software according to the user requirements; hence, we aim to apply this access type to our software. Normally the distribution, change, and application of the software is exclusive to the people that have the copyrights of the system, but with the open license all these characteristics are public for the users, for any use and purpose. Since our goal is to expand information, case studies, examples regarding our system, and, more importantly, validate its relevance, making it open access is a way to enable the accomplishment of this goal through the real-life implementation of our software. Additionally, it’s a way to enable the users to continue expanding and optimizing the software itself. If the system wasn’t made available via open access license, its modularity and implementation would be hindered and limited for the users, which goes against our purpose and beliefs. We aim to create a format of operation that constantly increases all kinds of possibilities of use, and, under the implementation of open access license, we are closer to it.

Reference: Lakhan, S. E., & Jhunjhunwala, K. (2008). Open source software in education. Educause Quarterly, 31(2), 32.

Our framework enables the possibility to solve conceptual issues across the scientific community based on sequential logic steps. The qualities and characteristics of our framework could help the improvement of emerging ideas and existing mechanisms by giving a new perspective of action for the solution of complex problems. Based on experts feedback, literature and iGEM project proposals we choose four areas to visualize the applicability and implementation for our framework and software that are described below.

Bioremediation

Polluted environments are normally contaminated with multiple toxic molecules, such as: pesticides, heavy metals and dangerous chemical compounds, generating a complex situation to remediate. Bioremediation on an uncontrolled system as a river or soil is difficult because every compound (either toxic or not) can affect the biological diversity and physicochemical properties of the environment. With our framework and visualizing this issue as a state machine, we can establish a solution model based on the bioremediation of specific compounds in certain timeframes to reduce the changes in the uncontrolled system and effectuate the remediation job in a more efficient way. It is also possible to construct a mechanism in only one chassis organism that could contain all the bioremediation system, without necessarily expressing it all at once, having the advantages of reducing costs and making the methodology simpler. Our framework could improve projects by simplifying their circuits, for example iGEM Hong Kong-City U 2020 aimed to make engineered bacteria that secrete multiple degradation enzymes to improve and make more efficient the plastic waste pollution. With sequential logic we can construct a system that expresses different degradation enzymes depending on the substrate and the specific moment. We talked to experts for feedback regarding the impact of sequential logic in bioremediation; Ing. Johan Morales Sánchez and Lic. Rossy Guillén Watson suggested that our system could be useful for waste-water treatment and for antibiotic traceability under different conditions. In addition, Lic. Victoria Zamora and Lic. Juan Carlos Hernández mentioned that bioremediation of multiple substances according to the present stimuli, environment and followed pathway could allow a remediation in the correct order to maintain balance and execute the task efficiently.

Biomedicine and Therapeutics

Diseases cause multiple metabolic and physiological reactions in the organism. If we visualize a disease as a state machine that changes through time with the biological responses of the organism, we can apply our framework to treat specific diseases in a more accurate way. Sequential logic allows us to treat diseases as a step by step issue, hence several solution pathways can be explored based on the state of development of the disease. Sequential logic could even aid the determination of a disease’s state, avoiding the use of invasive methodologies and improving the biomedicine field using sequential logic and synthetic biology. Our framework could also improve ideas and projects in the biomedical field like iGEM Nottingham 2020’s project. They aimed to make a biotherapeutic strain of Clostridium sporogenes to prevent, detect and treat neurodegeneration in patients. With our framework we can propose a system to know the state of neurodegeneration and make a personalized treatment depending on the development of the disease, complementing the iGEM Nottingham 2020 idea. When discussing this issue with experts they saw a relevant applicability of our framework to biomedicine and therapeutic aspects. Lic. Kendall Alfaro Jiménez and PhD. Carolina Centeno Cerdas suggested that our system could be useful to know when it’s necessary to change a treatment, such as chemotherapy or immunotherapy, and to establish diseases as a state machine in order to develop decision pathways to treat a certain sickness according to its real progression. Additionally, Ph.D. Candidate Kattia Nuñez Montero and PhD. Rodrigo Mora Rodríguez mentioned that with this system it would be possible to exchange a bacteria consortia to a single bacteria with multiple sequential capabilities, to develop an excellent screening system for different behaviors or pathways, and circuits that respond to different stimuli and follow diverse routes depending on the specific interest. This could help with diseases with inherent heterogeneity, such as cancer, allowing the exclusion of normal cells while covering the different subpopulations. Also, activating the therapy under specific conditions improves its specificity. These could be possible applications for the development of our framework in the biomedical and therapeutics field.

Industry

Every product of an industrial process needs to follow certain steps or states to produce the expected result. By visualizing industrial systems as state mechanisms, our framework and the sequential logic itself can be applied into the industry to optimize the paths followed for production according to specific requirements, products, methods and user needs. It could be possible to explore different pathways to produce a specific product and determine which pathway is the more efficient one. This could be achieved by making decision patterns that reduce metabolic burden while using metabolic engineering to reduce costs and improve methodologies of production and develop new mechanisms for the synthesis of certain compounds that are too complex or too expensive to produce. Our framework could be applied to characterize new methodologies and mechanisms of production, like the project from iGEM BIT-China 2020. Their idea was focused on the production of a co-culture of E.coli and S.cerevisiae to improve the fermentation industry of a specific vegetal compound (eriodictyol) with medical value. Our framework could be implemented to optimize the functionality of the system for the effective production of this molecule by establishing more efficient pathways of development and selecting the desired characteristics to applied into the circuit. Experts also told us that our framework could be really useful for industrial processes; PhD. Monserrat Jarquín Cordero, PhD. Pablo Jiménez Bonilla and PhD. Stefany Solano González suggested that our system could be functional for studying different pathways in just one circuit, determining the interaction of specific synthetic compounds with its sub-products, inducing production after a certain time and for verifying the functionality of a specific process. In addition, Bach. Laura Chaves Martínez, Ph.D. Candidate Olman Gómez Espinoza and M.Ed. Carolina Sancho Blanco mentioned that the formulation and development of nanoparticles, opening a path and segmentation for large scale metabolic engineering and determining the production and interaction of an expected product with its subproducts are potential applications for our system across the industry.

For our framework and software we define four areas of interest to focus our potential end users based on the applications we mention above. The four areas are the same as the circuit’s, but this section has a whole new perspective and interaction. First we select industrial stakeholders, the industrial area can be easily applied to sequential logic based systems due to its operation processes for the development of different products. Next we select bioremediation stakeholders that include bioremediation companies and environmentally focused non-governmental organizations. The same principles apply to bioremediation companies and biomedical stakeholders including the pharmaceutical industry. We picked gubernamental stakeholders: national biotechnology laboratories, public universities and national ministries. Gubernamental users have the power to start applying our system in a real setting . This would help us to introduce the implementation of the framework and software to start employing it as a new tool for the resolution of complex scenarios. Computation and bioinformatics users as well as researchers focused on investigation are important groups to consider for the introduction of the conceptual framework and software framework. Due to the computational understanding of these groups they could see a pioneering idea to expand this field of action. M. Sc. Raúl Trejos Espinoza mentioned to us that doing this project is great and a big challenge itself and can be a foundational work for projects and similar systems to come. In addition to this, companies that focus on the development of softwares and computational systems may be interested in our system in order to expand its catalogue and generate similar frameworks and softwares for different uses. On the other hand, policy makers who regulate the application of our technology are a crucial group to dialogue with due to the all potential applications that our system could be involved with. It's necessary to include these stakeholders to establish the correct development, regulations and conditions for the expected implementation of the project.

Stakeholder Map

We made a stakeholder map using the virtual tool “iGEMers Guide to the Future” (https://live.flatland.agency/12290417/rathenau-igem/) developed by a collaboration between iGEM teams and the European Mobilisation and Mutual Learning Action Platform SYNENERGENE. In this map we see our chosen stakeholders in a graph in which the x-axis means the interest of the stakeholder in our project and the y-axis means the power of the stakeholder to influence our project in a positive or negative way.

By making the stakeholder map and its analysis, we find that a great option to begin the implementation of our framework and software would be in collaboration with computational-bioinformatics experts and software companies. These stakeholders have the learning, experience, tools and interest to start applying the framework and software into test studies, new developments and even products. When the implementation of the system has been more structured, we aim to focus on the industry stakeholders. Our system could be easily adapted into industrial applications to diminish costs of production and for optimizing complex fabrication processes. For biomedical and bioremediation stakeholders, we expect to work with them when we expand data and results of our framework-software into the database, at that moment we expect to already have protocols, guides and models for different uses to start applying it into new models of resolution in these areas. For the regulation aspects, we aim to discuss with the policy makers to show them the functionality and applicability of our system to create the correct development route and conditions of use of the framework-software hand to hand with them. Actions have to be taken to guide the expected proposal of implementation for our system, but with dialogue between the different groups of stakeholders it should be possible to start including our technology within the real world. But in its first stage of introduction we aim to highlight the utility and applicability of the system to target groups to start developing the full potential of the conceptual framework and software framework.

Impact on scientific community

In the same way as our suicide circuit, we want the framework-software to have a real repercussion across the scientific community. Due to its capacities and fields of application mentioned above, we really see that our framework-software has the potential to be a novel and great tool that could expand the toolbox of the scientific community. This system can open many doors across the scientific community to make the construction of genetic circuits more practical, efficient and accurate. We expect that in its most structured form the software will be applied in any field of interest, to add another option in the workflow for the resolution of complex problems besides combinatorial logic based systems. Hopefully we expand the use of sequential logic based systems to explore new mechanisms, possibilities and emerging ideas that can be achieved by frameworks, softwares and circuits like ours.

Future approaches

To further explore more possibilities and options for our software framework, in addition with expert feedback, our software could be applied to gene-regulatory applications, enabling the determination of necessary gene expression, along with its time frame, for specific tasks. The software framework and sequential logic could be useful in the metabolomics area by having the possibility to explore different biosynthetic routes in a biochemical model and obtain information about certain metabolic processes. Additionally, multiple pathways and test studies could be incorporated in just one transformation process and, depending on specific inputs and the order in which they’re applied, the response (gene expression) could have endless variations. Other potential applications for the software framework could be evolutionary studies developed from the tracking of evolutionary trajectories by single cell sequentiation; it could also be possible to differentially tag cells depending on the state of the system according to PhD. Pablo Bolaños and PhD. Rodrigo Mora.

Future snapshots

For our framework and software we made a future snapshots analysis to preview the use and application of our system in a hypothetical future in which the technology is completely implemented. First, for our conceptual framework we expect the development of higher levels of specificity of its use and expand its approach to more complex biological systems. We visualize it as a standardized tool, an option that becomes part of the established mechanisms for the resolution of target problems. Furthermore, we expect an expansion in the use examples for each application area and case studies regarding the user requirements for its most efficient use. In addition, we want the conceptual framework to be a guide that enables the use and comparison between different potential frameworks to come, to obtain the correct perspective of the solution to target a complex problem. For our software framework we anticipate the use and extension of the generated library where the users consult it for a wide range of applications and include their data. We visualize the tool to be contemplated inside the basic toolbox for the analysis of these challenging scenarios; and to be linked to databases and more analysis tools for a bigger capacity of assessment in specific cases. Moreover, we look forward to the optimization of the software framework for cluster processing, that includes its code licensing to standardize the criteria and parameters for the evaluation of genetic circuits. The implementation of more complex analysis filters could be developed to expand inclusive more the applications of the system. We want to make an impact in coming projects and ideas to improve the scientific community and society.

Team:TEC COSTA RICA/Implementation

Implementation

This section aims to contextualize the circuit and framework for their application in multiple areas and conditions, in addition to expanding on what that would entail.

Context

Purpose

How are we different?

Why is it effective?

Advantages of our technology in the real world

Relevance of genetic counter circuits

Bioremediation

Biomedicine and Therapeutics

Industry

Biosensing

Risk Assessment

Risk Assessment

Circuits Malfunction

Environmental interactions

Bioterrorism

Dual-use implementation

SWOT: Strengths Weaknesses Opportunities Threats

Strengths

Weaknesses

Opportunities

Threats

How we are targeting those threats

Regulations

Stakeholder map

Impact on scientific community

Future approaches

Future snapshots

Context

Purpose

How are we different?

Why is it effective?

Advantages of our technology in the real world- Changing your mindset

Open access

Bioremediation

Biomedicine and Therapeutics

Industry

Biosensing

Strengths

Framework

Software

Weaknesses

Framework

Software

Opportunities

Framework

Software

Threats

Framework

Software

How we are targeting those threats

subtitle

Stakeholder Map

Impact on scientific community

Future approaches

Future snapshots