Team:Leiden/Entrepreneurship

<!DOCTYPE html> DOPLLOCK iGEM Leiden

Entrepreneurship

In our project, we propose and design DOPL LOCK, a system that is implemented to provide biocontainment for non- and semi-contained applications of GMOs. For our entrepreneurship program, the goal was to demonstrate our path to the implementation and commercialization of our DOPL LOCK system. This process includes the identification of the intended applications of DOPL LOCK and the implications of this implementation. Furthermore, we also describe the future developments and required capabilities to realize our system.

Executive summary

Firstly, we have identified that many non- and semi-contained GMO applications would benefit greatly from the implementation of DOPL LOCK, including wastewater treatment, bioremediation and whole-cell biosensors. Next, we have demonstrated that DOPL LOCK fulfills these unmet needs and specifically improves on existing biocontainment methods in the induction of lethality, the prevention of horizontal gene transfer and simplicity to implement.

Furthermore, we have concluded that our solution is both possible and scalable. This followed from our product design, based on the SEVA plasmids, that allows the incorporation of the described application and the adjustment of the standardized system to the prevalent conditions. Lastly, the final DOPL LOCK system is inventive, since it is not matched by previous developments and we have demonstrably improved on functionality, reliability and applicability of existing systems.

Additionally, we have envisioned that the long-term effects of the implementation of DOPL LOCK include enabling and stimulating various applications of GMOs. As a result, many developed solutions and valuable processes become closer to realization and can help to solve the most urgent global problems , such as climate change, pollution and non-sustainable food production. On the other hand, we have identified several negative effects that might arise with the application of DOPL LOCK, including resilience with part of the general public and various ethical considerations.

Moreover, we have drafted a comprehensive Safe-by-Design approach for the implementation of DOPL LOCK and this gave rise to the conclusion that the application of DOPL LOCK requires knowledge on the environmental risks, tailoring of the DOPL LOCK system and the license application. After extensive research and consultation with the responsible institutions, we have concluded that we have sufficient capabilities to perform these tasks, except for the in-depth legal understanding. Also, the general public, industry and regulators were identified as the most important involved stakeholders and future steps were formulated to convince them of the biosafety provided by DOPL LOCK.

Lastly, we have drafted a plan for the future steps of the realization of DOPL LOCK, which included stages of research and development, commercialization and regulatory consultation. Specifically, these described developments will presumably give rise to our finalized product and the capability to exploit DOPL LOCK as a start-up in 5 years. Furthermore, different sources of the required funding were identified and several risks were formulated for these developments.

  • Potential customers and unmet needs

    We proposed and designed DOPL LOCK, a system implemented to both contain genetically modified organisms (GMOs) at a specified area and also prevent the transfer of their genes. With our system, we aim to provide biocontainment for GMOs, thereby preventing the spread of synthetic organisms and genes in nature. As the use of GMOs is often associated with risks, such as losses of biodiversity and toxicity [1], it is crucial that proper biocontainment strategies are in place to prevent such consequences [2]. This need is especially apparent in the case of the non-contained use of GMOs, since there are no physical barriers to contain these organisms [3]. The existence of semi-contained GMO applications was introduced through our Human practices. These are applications in the environment where localised containment is possible, yet with a clear physical path for GMOs to spread into the environment. In these cases, it is crucial that proper biocontainment strategies are in place to mitigate the possible risk of this release. Specifically, some of the concerns include that the escaped GMO has a higher fitness than the naturally occurring microorganisms or that the synthetic activity could cause adverse effects [1]. One example is the spread of antibiotic resistance genes through horizontal gene transfer, thereby contributing to the global medical crisis of multi-drug resistant bacteria [4]. The aim of DOPL LOCK is to prevent these potential adverse effects, even for the non- and semi-contained application of GMOs. For our entrepreneurship program, we have therefore identified several areas of industry with a large potential for these applications that could tremendously benefit from this open-source DOPL LOCK solution.

    Wastewater treatment

    Many wastewater treatment plants use micro-organisms for the degradation of certain biodegradable compounds [5]. However, there are certain limitations to the use of these naturally occurring microorganisms. Firstly, the efficiency of these degradation processes is for some cases very low [6]. Consequently, it is difficult and time-consuming to get rid of all of these compounds entirely. Secondly, from our Human practices followed that various newly introduced and foreign substances cannot be degraded by naturally occurring microorganisms and accumulate. Besides, the range of non-degradable compounds is expected to only increase in the future. Therefore, we propose the use of GMOs as a solution to these challenges and enable efficient degradation of certain chemicals in wastewater treatment [6]. Bacteria can be engineered to improve or to enable a certain degradation pathway. With this, GMOs could degrade a much larger array of pollutants and also improve the efficiency of these processes when implemented in wastewater treatment.

    Bioremediation

    During the process of bioremediation, environmental contaminants are removed by the process of microbial degradation [7]. This degradation can occur through natural metabolic pathways or the application of GMOs with enhanced properties. Similar to the issue faced by wastewater treatment plants, the problem is associated with substances that are not (efficiently) removed from the environment and thereby accumulate over time. Therefore, the usage of GMOs could provide a way to more efficiently degrade a larger variety of different compounds [8].

    An example of the problems associated with these currently undegradable compounds is PFAS. Specifically, this compound is very stable and no naturally occurring microbe is able to degrade it [9]. As a result, PFAS accumulates in the environment and polluted sites are ubiquitous all over the world [10]. Previous work has been done to design a GMO that can degrade PFAS and potentially therefore solve this problem of environmental pollution [18]. With this result, we have performed a case-study to demonstrate that the application of DOPL LOCK enables the release of this GMO, which can be applied in bioremediation to remove excessive PFAS from the environment.

    Whole-cell biosensors

    Whole-cell biosensors consist of an engineered microbial platform that allows for the detection of a certain molecule [11], providing a field-based and cheap way of detecting, without the need of elaborate electronics and training [12]. Unfortunately, the application of these whole-cell biosensors is hindered by the concern for biosafety regarding the use of these GMOs [13]. This concern in fact prevents the development of these sensors, since the described applications of whole-cell biosensors all include the real-time monitoring of the environment, people and food [11]. We believe that DOPL LOCK can provide the required means of biosafety, by containing the GMO and the transformed genetic material. With this, we aim to take away this safety concern for the application of these biosensors and thereby bring them closer to realisation.

    iGEM projects

    Another considerable field that we believe could benefit from the implementation of DOPL LOCK is the iGEM competition. The results of this competition often indicate that synthetic biology could play an important role in solving some of the most urgent issues, such as climate change, pollution and non-sustainable food production. Many of these impactful iGEM projects also include non-contained applications of GMOs, which are in a lot of cases restricted or prohibited [14]. In fact, the iGEM competition rightfully has a 'Do Not Release Policy' due to the risks associated with the application of GMOs [15]. So, a lack of biosafety prevents the implementation of many of these ingenious iGEM projects. We have also concluded from our Human practices that many iGEM teams do not include biosafety for the reason of too much complexity and limited time. For these reasons, we believe that an easy-to-implement, reliable biocontainment system would be highly valuable to bring these solutions further towards realisation.

    To stress the variety and urgency of these non-contained synthetic biology applications, different examples are discussed. The first example of a developed synthetic biology solution was the iGEM team of Yale 2018 to target the problem of plastic accumulation, by introducing the recombinant enzyme PETase in a micro-organism [16]. Secondly, a solution was conceptualized for the nitrogen pollution and the consequent eutrophication by the 2016 iGEM team of Nebraska with environmental bioremediation [17]. The last example of an iGEM project of USAFA 2020 included the degradation of PFAS to prevent environmental pollution [18]. These solutions all target different compounds or molecules that are present in the environment, namely microplastics, nitrogen-compounds and PFAS respectively. For this reason, it is essential that proper risk assessments and biocontainment measures are in place, for the purpose of realising these beneficial solutions.

    Alternative biocontainment solutions

    In order to address these previously discussed opportunities, different types of biocontainment methods have already been conceptualised in previous research, which includes kill switches, auxotrophy and dependency-based biocontainment systems. In order to analyse the sufficiency of these methods for the proposed implementation, the specific advantages and drawbacks have been listed in table 1.

    Table 1: The benefits and drawbacks of different developed biocontainment methods

    Biocontainment method Benefits Drawbacks
    Kill switch Generic kill switch - Causes immediate death of the organism
    - Easily implemented inside the cell
    - Fast intervening    		 - Does not prevent the spread of the modified DNA
    - Requires human monitoring
    - Little control over the spread of the organism
    - Easily circumvented by mutations [19]
    Autonomous kill switch - Predefines the conditions in which the organism is able to survive
    - Easily implemented inside the cell
    - Does not require human monitoring    		 - Does not prevent the spread of the modified DNA
    - Easily circumvented by mutations
    Auxotrophy Auxotrophy to a particular cellular compound - Predefines the conditions in which the organism is able to survive
    - Easy defining of the boundaries    		 - Does not prevent the spread of the modified DNA
    - Delayed death of the organism after leaving the conditions [20]
    - No possibility to immediately intervene
    - Is easily circumvented by horizontal gene transfer of the missing genetic material or the exogenous production [21]
    Xenobiology-based containment - Predefines the conditions in which the organism is able to survive
    - The boundaries of release are easily defined
    - No circumvention with of the system through horizontal gene transfer of the missing genetic material    		 - Does not prevent the spread of the modified DNA
    - Delayed death of the organism after leaving the conditions
    - No possibility to immediately intervene
    - System is not modular
    - Difficult to implement [21]
    Dependancy-based biocontainment Geneguard - Prevents the transfer of genetic material
    - Multiple layers of safety [22]    		 - Does not prevent spread of the organism
    - Difficult to implement due to genomic integration requirement

    Generic kill switch: By introducing a kill switch, lethality is actively induced in the engineered organism after one or more conditions are specifically altered [20]. These conditions are imposed by humans when survival of the organism is no longer desirable.

    Autonomous kill switches: Through the functionality of an autonomous kill switch, lethality is actively induced in the targeted organism as a result of a change in environmental conditions [23]. This system is used for biocontainment when the conditions are defined to actively kill the cell when it leaves the defined conditions.

    Auxotrophy to a particular cellular compound: By introducing auxotrophy, the organism is not capable of synthesizing a certain cellular compound, which is necessary for its survival [20]. Instead, this compound is then supplied in the medium where it is supposed to survive. However, when the organism leaves this area, the essential compound will consequently not be present, which causes the organism to die. Therefore, this system can specifically be used for biocontainment, since the conditions and specific area for the survival of the organism are determined.

    Xenobiology-based containment: With this system, the organism is dependent on a synthetic molecule for its survival that has to be provided in the medium [24]. This could for example be achieved by requiring the cell to incorporate a non-standard amino-acid in some essential proteins.

    Geneguard: With the geneguard system, three independent mechanisms are employed to create a dependency between the host and the transformed plasmids [22]. The first mechanism includes the conditional origins of replication on the plasmids, for which the responsible factor is located on the genome. Secondly, auxotrophy is induced in the host by knocking-out the thymidine gene and supplying a copy of this on the plasmid. Lastly, an antitoxin is supplied in the genome, in order to counteract the activity of the toxin. The consequence of these mechanisms is that the plasmids cannot be separated from the host and this therefore serves as a way to counteract horizontal gene transfer.

    From table 1 firstly follows that a clear distinction can be made between biocontainment methods that prevent the spread of either the organism itself or its genetic material. The former goal is reached with both the kill switch and auxotrophy, thereby causing the organism to survive only within the specified conditions. On the other hand, the containment of the genetic material is reached with the dependency-based biocontainment systems, where it is prevented that the plasmids leave the organism.

    DOPL LOCK presents a unique solution that combines the functionality of both these biocontainment methods. This characteristic is very important, since the spread of the organism as well as the genetic material could potentially cause the risks associated with GMOs [26]. Moreover, the design of DOPL LOCK enables the integration of these containment strategies, thereby ensuring a compact design of the transformed genetic material.

    Furthermore, the application of DOPL LOCK allows for both the prevention of the spread of the organism as well as the capability to induce lethality. This is an important factor to be used in different scenarios and combines the functionality of the generic and the autonomous kill switch. This also allows the user to intervene in the process of the GMO and immediately trigger the induction of cell death, besides the definition of the system's boundaries.

    The last clear benefit of the use of DOPL LOCK over other similar biocontainment strategies is its simplicity to implement. This originates from the fact that no genomic integration or complex factors are required for the implementation of the system. This also facilitates the beneficial applicability and modularity of using DOPL LOCK. From our Human practices also followed that this simplicity to implement is crucial for developing a widely used biocontainment system.

    In order to also establish the unmet needs that DOPL LOCK could fulfill, we lastly conclude by summarising the benefits compared to the alternative biocontainment methods. The results of this analysis are represented in table 2.

    Table 2: Conclusions on the benefits of different biocontainment methods

    Biocontainment method Simplicity to implement Prevents HGT Prevents spread of the organism Immediate death after induction
    Generic kill switches :heavy_check_mark: :x: :x: :heavy_check_mark:
    Autonomous kill switches :heavy_check_mark: :x: :heavy_check_mark: :heavy_check_mark:
    Auxotrophy :x: :x: :heavy_check_mark: :x:
    Xenobiology-based containment :x: :x: :heavy_check_mark: :x:
    Geneguard :x: :heavy_check_mark: :x: :x:
    DOPL LOCK :heavy_check_mark: :heavy_check_mark: :heavy_check_mark: :heavy_check_mark:

  • Feasibility, scalability and inventivity

    Feasibility: Implementation of DOPL LOCK

    We have created a design for our final product that can be implemented in the wide range of described potential applications. Simple incorporation of DOPL LOCK would require an easy way of cloning and the facilitated tailoring of the system to the specific required conditions and restraints of the application. Therefore, we have firstly included a multiple cloning site inside the final double plasmid system, allowing for the ligation of the desired genes into the backbone we provide in our final product. We also made sure this multiple cloning site is biobrick compatible, allowing other iGEM teams to easily make use of our system.

    For the purpose of tailoring the DOPL LOCK system to the environmental conditions, we have used a modular system according to the Standard European Vector Architecture (SEVA) design. The SEVA backbone allows for a standardized backbone with the ability for modular reconstruction of the plasmids to specifically tailor the DOPL LOCK system to each application. Different inducible promoters and A/T systems are easily exchangeable and are used to enable the application of DOPL LOCK for the different specified conditions. In these SEVA plasmids of the DOPL LOCK system, the oriT is replaced by our own customly constructed biosafety module. This biosafety module includes a sequence that encompasses the inducible/constitutive promoter - toxin and the inducible/constitutive promoter - antitoxin. These modules are designed and constructed through the biobrick system. Thereafter, they are readily introduced in the backbone and construct the tailored DOPL LOCK system.

    Lastly, a consideration for the plasmid design was to build in a selection marker. This is essential to make a distinction between the cells that have taken up both or none of the plasmids after transformation. In our final system, the plan is to include a part of the split GFP selection marker on both plasmids, as is also described in the future developments [25]. This allows to visualize the complete fluorescence in the cells that have taken up both plasmids.

    Considering these different aspects of the product design, the final construction of the DOPL LOCK system is represented in figure 1.

    Placeholder

    Figure 1: Construction and architecture of the final DOPL LOCK system. The figure shows a SEVA 3.1 backbone and the proposed adjustments to create one of the plasmids for the DOPL LOCK system.

    Feasibility: regulatory restraints

    The feasibility of the application of DOPL LOCK is also determined by the current state of the regulations surrounding the use of GMOs. Specifically, this indicates the possibility of the non-contained application of GMOs in the intended processes described in potential customers. The regulations surrounding the non-contained use of GMOs are covered by the European Union (EU) Directive 2001/18/EC [27]. We designed an outline of this directive to show the possibilities and conditions for the non-contained application of GMOs. From this regulatory roadmap, the conclusions include:

    1. The application of non-contained GMOs is in fact possible throughout the European Union, following a license application and allocation from the responsible regulatory institution.
    2. For each application, an environmental risk assessment (ERA) has to be performed, for the purpose of evaluating the potential risks to human health and the environment.
    3. The final license application has to include the specified information for the execution of the ERA, the ERA and the conclusions from the ERA.

    From this also follows that the application of DOPL LOCK is possible on these non-contained applications. Moreover, these regulations also further emphasize the need for the proposed system to ensure the biosafety of the proposed solution. The requirements of the license application namely specifically state to provide information on [27]:

    1. Likelihood of the GMO to become persistent and invasive in natural habitats under the conditions of the proposed release(s).
    2. Potential for gene transfer to other species under conditions of the proposed release of the GMO and any selective advantage or disadvantage conferred to those species.

    In conclusion, these regulations indicate bio-safety conditions that align with the core of DOPL LOCK. These regulations thus also validate the necessity of systems like DOPL LOCK in order to ensure the biosafety of the proposed implementations.

    Feasibility: Consultancy

    In order to fully realize the implementation of DOPL LOCK, our plan is also to provide assessment and guidance in the license application. This followed from the fact that the realization of the application is fully dependent on the regulatory validation of the case-specific conditions and safety measures. Therefore, our approach for the implementation of DOPL LOCK consists of both the incorporation of biosafety as well as obtaining the subsequent regulatory approval.

    As a result of this approach, we also plan to incorporate a complete Safe-by-Design solution into these processes. During our Human practices, we discovered that the concept behind Safe-by-Design is to think about the possible risks at an early stage of the design of a new technical implementation. In our minds, this approach is very fitting for the incorporation of DOPL LOCK, because this firstly already requires a case-by-case approach of all the different aspects of biosafety. Secondly, the implementation of DOPL LOCK also includes the entire timespan of the risk assessment, incorporation of the system and the license application. Hereby, it is also crucial to already start thinking about biosafety from the start of developing a new process with the non-contained use of GMOs, because these considerations are essential for the approval of the license.

    The significance of providing this regulatory consultancy also reaches beyond our customers since we believe this will play a vital role in solving the established responsibility gap. Specifically, this gap consists of the absence of a responsible party, causing a vicious cycle of the regulators, scientists and the industry that hinders the development of new mature semi-contained GMO applications. We aim to solve this with our approach of both (i) addressing prevalent biosafety concerns of the industry with a standardized system as well as (ii) providing the legal basis for subsequently obtaining a license. This legal foundation is important to bridge the existing gap between industry and regulators and make the process of this first license application more attainable. With this, we believe that the industry finally has the required tools to realize the first semi-contained GMO applications and consequently also stimulate other parties in committing to similar goals. Hereby, we aim to bridge this disadvantageous gap that stands in the way of progress in the field of synthetic biology.

    Figure 2: Representation of DOPL LOCK as the bridge in the responsibility gap.

    Feasibility: Viability

    The demand in our provided final product and service also substantiated the feasibility of our system. This was firstly validated through our human practices by a representative from a company in wastewater treatment, Robert Wagenveld, who saw no issues with the implementation of our solution based on our PFAS case-study in the future. Secondly, the iGEM team of MIT_MAHE also agreed to implement DOPL LOCK for biosafety if they continue with commercially developing their project. In addition, we also concluded that there would be an interest in regulatory guidance we are providing for the license application. During our Human practices it was concluded that currently, a lot of companies are reluctant to apply for a license for a non-contained GMO application because of the necessary time and money investment. However, we believe that this process could be less daunting when it can be outsourced to a specialized company.

    Furthermore, the feasibility of DOPL LOCK is also influenced by the fact that the fully realized plans would come down to a pioneering position in the biocontainment field. Specifically, we have concluded that there are currently no similar companies that sell a complete Safe-by-Design tailored biocontainment solution for non-contained GMOs. The commercialisation of DOPL LOCK would therefore ensure this profitable market position without competitors. Lastly, it has to be noted that DOPL LOCK is intended to also function in stimulating the development of new synthetic biology solutions, as described in the long-term impacts. Therefore, more of these synthetic biology solutions would be conceptualized in the future and this results in an increasing market for biocontainment systems.

    Scalability

    The scalability of the solution of DOPL LOCK arises from the modularity of the designed product. This is important because all the ranging applications can be easily realized, even though this requires a case-by-case analysis of the specific conditions and tailoring of DOPL LOCK to the application. So, by making the system modular, we can ensure the scalability for the application in the wide range of described intended processes.

    A second important aspect of DOPL LOCK's scalability, is that we aim to provide a starting point for standardisation. Specifically, DOPL LOCK entails a modular platform for the biocontainment of a GMO, of which the gross structure of the double plasmids can be universally applied. Therefore, we want to build further on DOPL LOCK to fully ensure the standardisation, both as a biocontainment platform and from a legal perspective, as also indicated in our future developments. This standardised platform would also greatly contribute to scalability because this allows for easy and universal implementation, thereby only slightly modifying the system and license application for the different cases.

    Inventivity

    The designed DOPL LOCK system is inventive, since the final product is not matched by any current or previous developments. To reach this goal, we had built forward on the double plasmid system that was proposed in the iGEM 2019 Singapore team with the project E.co LIVE [28]. They developed these plasmids with a double toxin antitoxin (TA) system to both function as a way to control cellular productivity and plasmid retention. Throughout our project, we tried to build forward on this idea to specifically improve the functionality, reliability and applicability. This resulted in the following inventive modifications:

    1. Integrating a kill switch. The system of DOPL LOCK aims to provide a complete solution for biocontainment. The retention of the GMO itself also has to be considered next to the prevention of horizontal gene transfer that was introduced by E.co LIVE [28]. We tackled this problem by placing the antitoxins under inducible promoters in our design, thereby indirectly specifying the conditions for the activity of the toxin through the presence of an inducer. Our inventivity is also shown by the fact that this kill switch is fully incorporated into the existing double plasmid system. This requires less additional genetic material to be added to the cells and hereby also reduces the risk of a mutation in either of the systems.
    2. Introducing the concept of genomic integration. To solidify our proposed biocontainment strategy, genomic integration of one of the plasmids was considered to make the system more robust. From our safety reflection, we concluded that there is still a chance that both plasmids are transferred simultaneously, thereby also transmitting both antitoxins. Our hypothesis was that the chance of transfer of both plasmids is minimised when one of the plasmids is incorporated into the genome. Unfortunately, we were unable to show this in the lab but we plan on exploring this possibility further in our future developments.
    3. Aiming the application to biocontainment for the non-contained use of GMOs. In our project, we fully developed the system to be applicable for the containment of GMOs and their modified DNA and built our entire project around this. This would enable a new purpose for the foundation of the double plasmid system and thereby also a new market.

  • Long-term effects

    Ultimate goal of DOPL LOCK

    With DOPL LOCK, our ultimate goal is to enable the use of non-contained GMOs for the purpose of solving both global and local urgent problems. By continuing to improve our system, we aim to optimize the biocontainment provided by DOPL LOCK and attempt to validate the safety through assays, as detailed in development plans. Following this, DOPL LOCK could serve as a standardized biosafety system. Consequently, many non-contained GMO applications are in this scenario enabled and these are often designed to tackle global problems, as, for example, shown by many projects within the iGEM competition. The variety and benefits of these processes are large, ranging from the remediation of microplastics [16] and PFAS [18] to the prevention of eutrophication [17].

    Indirect consequences of DOPL LOCK

    Furthermore, we believe that an indirect consequence of the realization of DOPL LOCK is the stimulation of the development of similar synthetic biology solutions. From our Human practices, we have concluded that the development of synthetic biology is currently decelerated due to the current regulatory restraints. Specifically, it is presently not advantageous or profitable to continue the development of such non-contained GMO applications. This situation is not desirable since this stands in the way of possible progress and developed solutions in this field. However, if DOPL LOCK proves to enable these similar processes, this would also provide perspective for these applications. Thus, we believe that DOPL LOCK will play an important role in unleashing the full potential of synthetic biology. In order to realize this, we have also decided to propose DOPL LOCK as an open-source solution. This will allow other ambitious researchers and iGEM team to make use of our provided biosafety and thereby stimulate these developments. Additionally, we believe that this will not threaten the existence of our startup, since this provides a lot of extra guidance that will help the realisation of the application.

    Potential negative consequences of DOPL LOCK

    A first possible negative impact that could arise with the application of DOPL LOCK is that the enabled synthetic biology processes could eventually lead to an uncontrolled outbreak of GMOs. This could potentially occur because the mechanism of DOPL LOCK is currently not completely failproof yet. Therefore, we have already performed an analysis of the safety concerns with the use of DOPL LOCK. Furthermore, the results from this risk assessment will be used to minimise the identified possible hazards, as described in the future developments.

    A second possible negative effect arises from a potential stimulation to the use of GMOs that pose a threat to human health or the environment. So far, we have mostly considered safe strains since the genes they (i) carry are not dangerous or (ii) clearly provide a selective advantage. Examples of these GMOs that are accompanied with more risks include: antibiotic resistance genes and expressed pesticides [26]. In this case, the potential hazard of the spread of these organisms increases, demanding more restraints on the applied biocontainment method. It is crucial that a risk assessment is to be executed and that the sufficiency of DOPL LOCK is evaluated.

    Thirdly, the application of these GMOs, as enabled by DOPL LOCK, might cause a strong resilience with a part of the public. A significant portion of the public is suspicious of the safety and the justification of the use of GMOs, which also followed from the results of our survey. This suspicion will presumably be increased when these GMOs are suddenly allowed to be used in a non-contained application.

    Ethical considerations

    Finally, to assess the long-term impacts of the implementation of DOPL LOCK, we have also examined the ethical considerations. Specifically, much discussion has focused around the justification of genetically modifying an organism. Possible objections for the use of GMOs firstly include that these modifications result in unnatural organisms, as is also mentioned in the European legislation [29]. This would oppose the organic and natural products that many people consider favourable. Another possible objection for the use of GMOs originates from the notion of dignity of the organism [30]. This means that people should not be allowed to manipulate other living organisms for their own purposes.

    The results of our survey also showed that a considerable number of people did not support the application of GMOs as a result of their ethical concerns. Specifically, on average, people indicated to consider the application of GMOs to be dangerous as a result of the ethical considerations with a score of 5.66 out of 10 (absolutely dangerous). However, previous research has concluded that the support of the use of GMOs increases when more scientific background is provided [31]. In addition, the results of the survey also demonstrated that about 75% of the respondents were more inclined to support the application of GMOs after knowledge of our system. For these reasons, we believe that more education about the meaning of GMO and our system will eventually result in more endorsement of the application of GMOs.

  • Skills, capabilities and stakeholders

    To analyse the necessary capabilities for the further development of our solution, we have identified the different aspects required for the implementation of DOPL LOCK. As previously mentioned, this implementation requires a Safe-by-Design approach. With this comprehensive approach, the evaluated aspects of the implementation are:

    1. Assessment of the environmental conditions and risks of the suggested application
    2. Implementation of DOPL LOCK into the suggested application on a scientific level
    3. Regulatory validation and license application for the developed system

    Next, we have established the implications of these different aspects, to thereby conclude on the required expertise and the involved stakeholders.

    Assessment of the environmental conditions and risks

    This first step is important to specifically tailor the DOPL LOCK system to the requirements and the conditions of the specific application. For this purpose, a risk assessment has to be conducted that identifies the main hazards of the introduction of the intended GMO. Secondly, information should be gathered on the conditions within the application and the potential boundaries of the system.

    This assessment requires knowledge of the ecology and environment of the intended application. We plan to conduct this by following the steps in the environmental risk assessment (ERA) as described in our regulatory roadmap. This tool was introduced by the EU as a way to identify the different potential risks with the introduction of GMOs within the environment and proposed management strategies. This ERA also forms the groundwork for the final license application and is, therefore, also an essential part of the implementation of DOPL LOCK. Following our intensive research and communication with the RIVM, COGEM and other regulatory bodies within our Human practices, we believe that we have sufficient knowledge within our team to perform these risk assessments. Examples of our exact execution of this ERA are provided with our case-studies.

    Design of the implementation of DOPL LOCK

    The next step in the application of DOPL LOCK is the tailoring of the system to the intended process. This tailoring is based on the risk assessment and the environmental conditions, which were concluded from the first step in this approach. With this, the DOPL LOCK system is modified to the intended boundaries and safety consideration of the application.

    The implementation, therefore, requires knowledge of science, biosafety and the modular design of DOPL LOCK. In order to make this process of tailoring DOPL LOCK easier, we have worked on a step-by-step approach of the plasmid design for our proposed implementation. With this, we provide a tool that can aid in the process of designing the implementation of the DOPL LOCK system. In addition, we have shown the specific design of DOPL LOCK to accommodate two practical implementations within our case-studies.

    Accomplish the regulatory validation and license application

    During the implementation of DOPL LOCK, regulatory validation must be obtained since this is essential to realize the desired application. In the license application, it has to be demonstrated that the proposed solution does not pose a threat to human health and the environment.

    This regulatory assessment requires in-depth knowledge about the current legislation surrounding the non-contained use of GMOs. To show our consideration of these regulatory restraints, we have created a roadmap to a license application for the non-contained use of GMOs. However, as our team consists of biotechnologists, we would want to collaborate with license experts when developing our solution and company further.

    Stakeholders

    With the described approach of the implementation of DOPL LOCK, different stakeholders have to be taken into account. For this purpose, the most important involved stakeholders were identified and represented in table 3.

    Table 3: Identification of the the primary stakeholders.

    Part of the DOPL LOCK approach Primary stakeholder Main interest
    Risk assessment Public/legislators Safety
    DOPL LOCK implementation Customers Efficiency of the intended process
    Regulatory validation Legislators Enforce the legislations

    During the steps of the risk assessment, we identify possible hazards in order to assure the safety of the society. The public is, therefore, an important stakeholder to take into account when performing these steps. We have conducted a survey to evaluate opinions of the public on the use of GMOs. From these results, we have concluded that approximately 75% of the respondents were more inclined to support the application of GMOs with knowledge of our system. Further public engagement and education is, therefore, needed in order to raise awareness and trust in the biosafety that DOPL LOCK would create.

    Another important stakeholder during the implementation of DOPL LOCK are the customers. The main interest of these stakeholders is obtaining the tailored biocontainment system in order to enable their proposed process. Additionally, the efficiency of the application has to be maintained, despite the introduction of this metabolically expensive DOPL LOCK and the imposed environmental conditions. To show this, more experiments have to be performed as also described in the future developments. With these experiments, we aim to further convince these potential customers of the feasibility of the combined DOPL LOCK system and the proposed application.

    Legislators also have to be taken into account, since the final approval of the suggested applications is their responsibility. As previously discussed, we plan to convince legislators by firstly assessing the safety of the application and following the proposed roadmap for the eventual license application. Furthermore, we would like to build forward on the standardisation of DOPL LOCK from a regulatory perspective. Further steps are described in the future developments to prove the general functionality and data has to be acquired on the overall provided biosafety. This would allow the validation of the system to process easier in the future, which is a beneficial step in the scaling up of DOPL LOCK.

  • Future developments

    During our project, we have established a plan for DOPL LOCK after the iGEM competition. This plan was based on the milestones that have to be achieved to reach the complete realization of DOPL LOCK and the consequent beginning of a startup. These milestones, therefore, represent the main stages of the future developments of DOPL LOCK and are reflected in figure 3.

    Milestones

    Figure 3: Formulation of the milestones for the realization of DOPL LOCK.

    According to this plan, DOPL LOCK would be fully developed and released into a complete business after 4-5 years of the described future developments. We have also identified each individual step within the timeframe that has to be executed to reach these goals. The results of these future developments and the approximate time requirements are represented in a Gantt chart, as displayed in figure 4.

    Gantt chart

    Figure 4: Gantt chart for the future developments of DOPL LOCK.

    These results show that the further development of DOPL LOCK includes phases of research and development, legislative consultation and the final commercialisation. Lab facilities and equipment are required to carry out these developments. Also trained staff is required in order to continue this research, which is composed of our qualified team.

    Another necessary resource for this entire business plan is funding. This was also described in the development plans and we aim on achieving this by both applying for funds and finding investors. Firstly, the developments of the early stages of our research will presumably depend on the funding obtained from grants. We have already identified several different sources of this funding, that will therefore be used to start-up the developments of DOPL LOCK. In the Netherlands, the main institutions that provide grants for the development of research projects include the Netherlands Organisation for Scientific Research (NWO), the Netherlands Enterprise Agency (RVO) and the Royal Netherlands Academy of Arts and Sciences (KNAW) [32]. Furthermore, we have looked into these organisations to identify potential grants that could be allocated for our early research phase of DOPL LOCK. Examples of these grants and sources of funding include the Take-off program and Open mind. These instances of grants specifically focus on societal application and impact of research, which specifically correspond to the foundation of the development of DOPL LOCK. With the application to these grants, we will kickstart the future developments up to the point of testing the system for its functionality.

    After finishing this phase of early developments, we aim to have proven the functionality and provided biosafety of DOPL LOCK. We believe that this will help to attract further investors that are interested in the future development of DOPL LOCK. This targeted group of investors mainly include companies in the described potential market of DOPL LOCK. With this new source of funding, we hope to fully realize the development of DOPL LOCK and the startup.

    During the conceptualisation of these plans, we also considered possible risks surrounding the developments of DOPL LOCK and the startup. Consequently, plans could be developed to anticipate and circumvent these possible hurdles for the development of DOPL LOCK. The most important identified risks include:

    1. undesirable results
    2. insufficient funding
    3. not meeting all regulatory restraints

    SWOT analysis

    As we have concluded from our milestones, DOPL LOCK could be turned into an existing company after 4-5 years of the described future developments. In order to grasp the position this resulting company might fulfill, we have already performed a SWOT analysis. The results of this analysis are represented in table 4.

    Table 4: SWOT analysis

    Strengths Weaknesses
    - Modular
    - Simple-to-implement
    - comprehensive biocontainment system
    - complete Safe-by-Design solution    		 - Further testing needed
    - No way to circumvent specific mutations
    - Tight regulatory restraints
    - Insufficient legislative knowledge
    Opportunities Threats
    - Synthetic biology has much future potential
    - Can take away a barrier of bureaucracy
    - High urgence due to current global issues
    - No current similar service    		 - Reluctance to enter the long process of a license approval- Fear of GMOs by the general public
    - Possible future competitors
    - Depending on grants for the early stage funding

References

  1. Prakash, D., Verma, S., Bhatia, R., & Tiwary, B. N. (2011). Risks and Precautions of Genetically Modified Organisms. ISRN Ecology, 2011, 1–13. https://doi.org/10.5402/2011/369573
  2. Lee, J. W., Chan, C. T. Y., Slomovic, S., & Collins, J. J. (2018). Next-generation biocontainment systems for engineered organisms. Nature Chemical Biology, 14(6), 530–537. https://doi.org/10.1038/s41589-018-0056-x
  3. Biosafety and the Environmental Uses of Micro-Organisms. (2015). Harmonisation of Regulatory Oversight in Biotechnology. Published. https://doi.org/10.1787/9789264213562-en
  4. Lerminiaux, N. A., & Cameron, A. D. (2019). Horizontal transfer of antibiotic resistance genes in clinical environments. Canadian Journal of Microbiology, 65(1), 34–44. https://doi.org/10.1139/cjm-2018-0275
  5. Food and Agriculture Organization (FAO)., Wastewater treatment and use in agriculture. FAO irrigation and drainage paper No. 47, 1992
  6. Nzila, A., Razzak, S., & Zhu, J. (2016). Bioaugmentation: An Emerging Strategy of Industrial Wastewater Treatment for Reuse and Discharge. International Journal of Environmental Research and Public Health, 13(9), 846. https://doi.org/10.3390/ijerph13090846
  7. Aquino, E., Barbieri, C., & Oller Nascimento, C. A. (2011). Engineering Bacteria for Bioremediation. Progress in Molecular and Environmental Bioengineering - From Analysis and Modeling to Technology Applications. Published. https://doi.org/10.5772/19546
  8. Kumar, N. M., Muthukumaran, C., Sharmila, G., & Gurunathan, B. (2017). Genetically Modified Organisms and Its Impact on the Enhancement of Bioremediation. Energy, Environment, and Sustainability, 53–76. https://doi.org/10.1007/978-981-10-7485-1_4
  9. Bolan, N., Sarkar, B., Yan, Y., Li, Q., Wijesekara, H., Kannan, K., Tsang, D. C., Schauerte, M., Bosch, J., Noll, H., Ok, Y. S., Scheckel, K., Kumpiene, J., Gobindlal, K., Kah, M., Sperry, J., Kirkham, M., Wang, H., Tsang, Y. F., . . . Rinklebe, J. (2021). Remediation of poly- and perfluoroalkyl substances (PFAS) contaminated soils – To mobilize or to immobilize or to degrade? Journal of Hazardous Materials, 401, 123892. https://doi.org/10.1016/j.jhazmat.2020.123892
  10. Brusseau, M. L., Anderson, R. H., & Guo, B. (2020). PFAS concentrations in soils: Background levels versus contaminated sites. Science of The Total Environment, 740, 140017. https://doi.org/10.1016/j.scitotenv.2020.140017
  11. Moraskie, M., Roshid, M. H. O., O'Connor, G., Dikici, E., Zingg, J. M., Deo, S., & Daunert, S. (2021). Microbial whole-cell biosensors: Current applications, challenges, and future perspectives. Biosensors and Bioelectronics, 191, 113359. https://doi.org/10.1016/j.bios.2021.113359
  12. Hicks, M., Bachmann, T. T., & Wang, B. (2019). Synthetic Biology Enables Programmable Cell‐Based Biosensors. ChemPhysChem, 21(2), 132–144. https://doi.org/10.1002/cphc.201900739
  13. Wan, X., Ho, T. Y. H., & Wang, B. (2019). Engineering Prokaryote Synthetic Biology Biosensors. Handbook of Cell Biosensors, 1–37. https://doi.org/10.1007/978-3-319-47405-2_131-1
  14. Davison, J., & Ammann, K. (2017). New GMO regulations for old: Determining a new future for EU crop biotechnology. GM Crops & Food, 8(1), 13–34. https://doi.org/10.1080/21645698.2017.1289305
  15. The iGEM foundation. (2021). Safety/Do not Release - 2021.igem.org. Do Not Release Policy. https://2021.igem.org/Safety/Do_not_Release
  16. Chang, K., Gao, C., Li, K., Moscoso, R., Tirad, A., & Telesz, L. (2018). Team:Yale/Description - 2018.igem.org. IGEM Yale. https://2018.igem.org/Team:Yale/Description
  17. Bierman, M., Dooling, D., Harper, C., Mueller, J., Ninh, P., & Schwabauer, B. (2016). 2016 Project International Genetically Engineered Machine Team Nebraska. IGEM UNL. https://igem.unl.edu/2016-project
  18. Till, A., Swicegood, B., Jeon, C., Lochmaier, P., Loesch, A., Harris, J., Doherty, M., Pham, T., Orahood, O., Walters, C., Holland, A., McHugh, E., Reicher, M., & Raab, M. (2020). Team:USAFA/Description - 2020.igem.org. IGEM USAFA. https://2020.igem.org/Team:USAFA/Description
  19. Wright, O., Stan, G. B., & Ellis, T. (2013). Building-in biosafety for synthetic biology. Microbiology, 159(Pt_7), 1221–1235. https://doi.org/10.1099/mic.0.066308-0
  20. Whitford, C. M., Dymek, S., Kerkhoff, D., März, C., Schmidt, O., Edich, M., Droste, J., Pucker, B., Rückert, C., & Kalinowski, J. (2018). Auxotrophy to Xeno-DNA: an exploration of combinatorial mechanisms for a high-fidelity biosafety system for synthetic biology applications. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0105-8
  21. Lee, J. W., Chan, C. T. Y., Slomovic, S., & Collins, J. J. (2018). Next-generation biocontainment systems for engineered organisms. Nature Chemical Biology, 14(6), 530–537. https://doi.org/10.1038/s41589-018-0056-x
  22. 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. https://doi.org/10.1021/sb500234s
  23. Caliando, B. J., & Voigt, C. A. (2015). Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nature Communications, 6(1). https://doi.org/10.1038/ncomms7989
  24. Kunjapur, A. M., Napolitano, M. G., Hysolli, E., Noguera, K., Appleton, E. M., Schubert, M. G., Jones, M. A., Iyer, S., Mandell, D. J., & Church, G. M. (2021). Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells. Science Advances, 7(27), eabf5851. https://doi.org/10.1126/sciadv.abf5851
  25. Romei, M. G., & Boxer, S. G. (2019). Split Green Fluorescent Proteins: Scope, Limitations, and Outlook. Annual review of biophysics, 48, 19–44. https://doi.org/10.1146/annurev-biophys-051013-022846
  26. Prakash, D., Verma, S., Bhatia, R., & Tiwary, B. N. (2011). Risks and Precautions of Genetically Modified Organisms. ISRN Ecology, 2011, 1–13. https://doi.org/10.5402/2011/369573
  27. Council directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC (2001) Official Journal
  28. Zhi, L. X., Xuan, E. W., Lim, J., Chan, S., Chunyang, S., Lim, C., Iyer, S., Suda, S., & Hao, Z. (2019). Team:NUS Singapore - 2019.igem.org. Ecolive. https://2019.igem.org/Team:NUS_Singapore
  29. Christiansen, A. T., Andersen, M. M., & Kappel, K. (2019). Are current EU policies on GMOs justified? Transgenic Research, 28(2), 267–286. https://doi.org/10.1007/s11248-019-00120-x
  30. Mayes, C. (2014, 26 juni). Because we can, does it mean we should? The ethics of GM foods. The Conversation. https://theconversation.com/because-we-can-does-it-mean-we-should-the-ethics-of-gm-foods-28141
  31. Diamond, E., Bernauer, T., & Mayer, F. (2020). Does providing scientific information affect climate change and GMO policy preferences of the mass public? Insights from survey experiments in Germany and the United States. Environmental Politics, 29(7), 1199–1218. https://doi.org/10.1080/09644016.2020.1740547
  32. Rathenau Institute. (2021, August 2ndt). Funding and performance of R&D in the Netherlands Rathenau Instituut. https://www.rathenau.nl/en/science-figures/investments/how-much-does-netherlands-spend-rd/funding-and-performance-rd

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