Team:Leiden/Implementation

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DOPL LOCK

Implementation

In our project, we propose and design DOPL LOCK, a system that can be implemented to provide biocontainment for non- and semi-contained GMO applications. We have identified several potential implementations for our system, including applications within iGEM and the industry sectors of wastewater treatment, bioremediation and whole-cell-biosensors. DOPL LOCK can cover this wide range of applications due to its modular design, in which the standardized SEVA design has been adapted to include modular cassettes. To realize the implementation of DOPL LOCK, several challenges have been identified and solutions are proposed to overcome these hurdles. Finally, we show the tailored implementation of DOPL LOCK in two use-cases: on a GMO that degrades poly- and perfluoroalkyl substances (PFAS) and one that is involved in higher yield agriculture.

End-users

With DOPL LOCK, we designed a system that can both contain an organism at a specified place and simultaneously prevent the transfer of synthetic genes to naturally occurring bacteria in the ecosystem. With this, we hope to provide a biocontainment strategy for using genetically modified organisms (GMOs) in non- and semi-contained applications (see the Entrepreneurship page for further elaboration on these applications). Currently, the risk of the GMO spreading still hampers and even completely blocks the use of many ingenious solutions to real-world problems that require the use of semi-contained GMOs. Since there are extensive risks involved with the spread of GMOs and/or their DNA, the process of gaining approval to use GMOs in a non- and semi-contained situation is restrained and/or unfeasible in most countries [1]. However, we believe that implementing DOPL LOCK to ensure the biocontainment of GMOs, would open up a world of possibilities to make progress in many different research areas and applicable in real-world scenarios.

These ingenious problem-solving GMOs include a wide range of applications for which we have considered the possible implementation of DOPL LOCK. This variety of different applications for GMOs is clearly demonstrated by the yearly results of the iGEM competition. Specifically, this proves that many contemporary global and local problems can be tackled by synthetic biology. The world could tremendously benefit from these solutions, if they could be realized. Examples include climate change (Team: 2016_Chalmers), pollution (Team: 2020_USAFA), and non-sustainable food production (Team: 2021_MIT_MAHE). Moreover, in our Human Practices we have identified fields of synthetic biology that have already looked into the possibility of using GMOs and could thus tremendously benefit from our system. These fields include bioremediation, wastewater treatment, whole-cell biosensors and desalination (see Entrepreneurship page for specific details). We believe that the implementation of DOPL LOCK in these processes could provide the tools to ensure biosafety, which would help gain approval to use the GMO in a non- and semi-contained situation to allow its function.

We have identified the end-users of DOPL LOCK to be scientists, start-ups, ambitious iGEM teams or even big industrial companies with an innovative, clever and practical idea to solve a global or local problem with GMOs outside of the laboratory. Their idea or product could be combined with DOPL LOCK to create a GMO that is both biosafe and able to perform its envisioned function. DOPL LOCK can be used for many applications, as DOPL LOCK is modular, personalizable and standardized, as enabled by our product design. With this, we aim to bring the incredibly large range of synthetic biology applications closer to realization. We believe that the full realization and implementation of DOPL LOCK will enable and stimulate the development of the non and semi-contained use of GMOs in a safe manner.

Implementation

Because we aim to facilitate a wide range of applications with our system, the main consideration for the product design was to make sure it is easily implemented to different applications. This requires both an easy way of cloning and the precise tailoring of the system to the conditions and restraints required for the specific application. Considering these aspects, the final DOPL LOCK system was designed as is represented in figure 1 (as is detailed in the Entrepreneurship page):

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.

As a result of this specific design, different synthetic elements are easily cloned into the system using a multiple cloning site (MCS). Furthermore, the interchangeable safety module allows for simple tailoring of the toxin antitoxin (TA) system to the conditions of the release. The final DOPL LOCK system is therefore standardized, but modular, personalizable and compatible with many different applications. The elements in our biocontainment system can, namely, easily be switched to accommodate the specific needs of the end-user. Additionally, the copy number amounts and GC content of the DNA can be optimized for each individual end-user.

Origin of replication (Ori)

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

Toxin

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

Promoter for the toxin

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

Promoter for the antitoxin

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

Antitoxin

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

Selection cassette

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

Cargo

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

Challenges

To make sure we will be able to work with researchers to achieve a Safe-by-Design GMO, some challenges still need to be addressed in the further development of our proposed system. Further details about these future developments can be found on the Entrepreneurship page.

Table 1: Challenges to be addressed in the further development plans

Challenge Proposed solution
Easy cloning of the system Optimizing PCR cloning or DNA synthesis
Balancing the plasmids Developing the model further
Preventing interference with cellular metabolism Decreasing the metabolic burden
Expanding the number of applications and host organisms of DOPL LOCK Testing of different TA systems and promoters
Proving of functionality Developing testing methods
Gaining approval from regulators and the public Educating and engaging with the regulators and public

Firstly, cloning of the system should be done in an easier way than the Restriction Enzyme Based Cloning, since the toxicity of the toxin interferes with the process. The toxin kills cells before they get a chance to divide and grow. Possibly, this could be solved with optimized PCR cloning or DNA synthesis [7]. Furthermore, these methods would allow us to specifically design the needed promoter and TA system combinations for each application.

Additionally, more time needs to be spent on ensuring the balance between the two plasmids in the developed system. If the system becomes unbalanced, the GMO will be killed by the toxin. Therefore, this does not pose a safety risk, but a practical one. This needs to be tackled to ensure this does not lower the efficiency of the GMO. First efforts have been made with our model, but this needs to be optimized further.

In addition to balancing the expression of the TA system, we need to ensure that the DOPL LOCK system interferes as little as possible with cellular metabolism. In particular, a sufficient amount of protein of interest should be produced with the DOPL LOCK system in place. Even though safety is always at the cost of production, this price should not be too high. By decreasing the metabolic burden of the DOPL LOCK, less interference will take place on the intended functionality of the GMO. Options to lower the metabolic burden include lowering the expression levels of the TA systems, making the toxin and antitoxin proteins smaller and top-down genome streamlining [8]. The lowering of the metabolic burden will result in a GMO with sufficient production of the protein of interest or sensing abilities, while DOPL LOCK is in place to ensure the safety of the organism.

Furthermore, the number of applications and host organisms DOPL LOCK is suitable for could be expanded from our proof of concept based on Escherichia coli. To achieve this, more options for applicable TA systems and usable promoters need to be tested. With more options for the TA systems, such as synthetic toxins and nucleases, as well as inducible promoters, such as non-toxic compounds, light- or pH-inducible promoters, we will be able to expand our system to more varied environments and to different host organisms. This will highly expand the range of potential applications that DOPL LOCK can be implemented in. Possibly, the system requires more adjustments to adapt it to additional host organisms, which we will determine with further research, as described in our future development plans.

Lastly, definitive and long term proof needs to be acquired on the safety and functionality of the DOPL LOCK system. To this end, we should develop specific testing methods that can definitively show the functionality of DOPL LOCK at a long term scale. Using this long term data, we can consult with regulators for the purpose of potentially accepting DOPL LOCK as a standard management system for GMOs in a non-contained setting.

Furthermore, using this proof and the acceptance of regulators we can further educate the general public on our system. With this educational program we hope to raise awareness of the potential GMOs have to save the world and gain their trust that we solve those global issues in a safe manner using DOPL LOCK.

Case-studies

To show some examples of the implementation of DOPL LOCK in specific applications, two case studies were explored. These examples show that the system is easily tailored to be incorporated into a wide range of suggested (synthetic biology) applications, making the system modular and personalizable.



PFAS case-study

This case study was performed to show the implementation of DOPL LOCK and the potential of a Per- and polyfluoroalkyl substances-degrading (PFAS-degrading) GMO that can be used in a semi-contained application. The motivation for this specific case is that PFAS pollution causes a global problem that is currently not or not efficiently solved by existing methods, as was indicated through our Human Practices. This firstly poses a problem to the field of wastewater treatment, since these substances cannot be efficiently removed from the polluted water. Secondly, this is also an environmental problem for the field of bioremediation because the persistent material PFAS accumulates in the environment. Therefore, there is huge potential for a PFAS-degrading GMO, which was also pointed out through our Human practices.

Poly- and perfluoroalkyl substances (PFAS) are a group of synthetic chemical compounds used in various materials [9]. These substances are highly resistant to thermal and chemical degradation [10]. Another difficulty in the degradation lies in the fact that no naturally occurring microbe was yet found that can degrade this foreign substance. These facts pose a serious problem for the environment since there is essentially no easy way to get rid of introduced PFAS through biological, chemical or thermal degradation.

Consequently, PFAS that is released into the environment is very hard to remove. This poses a serious issue, as it has been demonstrated that this pollution causes a serious threat to both human health and the environment [11]. Nevertheless, the pollution of PFAS into the environment is widespread, for example counting about 2,337 contaminated sites across the united states [12]. It is therefore crucial that methods are developed for the efficient removal of PFAS from the environment.

For this purpose, different methods were developed, which include granular activated carbon and reverse osmosis. The problem with these techniques is that they are expensive and difficult to implement. Specifically, these techniques still produce PFAS-containing compounds that still have to be dealt with and do not fully solve the problem [13].

Currently, there is still no fully-developed, cheap and effective way to remove PFAS from the environment [13]. Existing methods operate by extraction of the substance from the environment, but thereby still produce PFAS that has to be dealt with, as discovered through our Human Practices. The solution could be provided by designing and implementing PFAS-degrading GMO. This idea was conceptualized by the iGEM USAFA 2020 team that tried to solve the problem of PFAS pollution. However, this valuable idea was never realized because there was no way to safely release GMOs into the environment, as stated on their wiki.

→ In conclusion, to enable this efficient way to degrade PFAS, a fully reliable biocontainment system has to be developed first.

Risks

To assess if our system would suffice in the context of a PFAS use case, we first identified the risks that are associated with a PFAS-degrading GMO without the implementation of DOPL LOCK. We based this risk assessment on the environmental risk assessment (ERA), as described in annex II of Directive 2001/18/EC and our own regulatory roadmap. This ERA also forms the basis for the license application for the approval of the non-contained use of GMOs (see Human Practices page for further details).

The steps in the ERA include:

  1. Identification of characteristics that may cause adverse effects
    1. The PFAS-degrading GMO will contain genes that are necessary for the dehalogenation of these compounds.
  2. Evaluation of the potential consequences of adverse effects
    1. The developed PFAS-degrading GMO may receive a selective advantage and could consequently outcompete naturally occurring microorganisms. The selective advantage could presumably originate from the obtained ability to use a new carbon source. This would lead to the gradual disappearance of other organisms without the capability to degrade PFAS and thus a loss of biodiversity.
    2. The GMO could have an undesired and harmful function if the GMO is spread to a site in which the presence of PFAS is desired. Specifically, PFAS is for example used in construction, glass and fire-extinguishers [9]. Spreading of the GMOs to PFAS-containing sites could lead to the gradual degradation and dysfunction of these materials.
    3. The spread of the synthetic genes to wild-type organisms. These organisms thereby receive the new characteristics, in which case the spread and consequences become more unpredictable. The transfer of this synthetic material could occur through horizontal gene transfer.
  3. Evaluation of the likelihood of the occurrence of each identified adverse effect
    1. The event of the loss of biodiversity is not very likely to occur since the degradation of PFAS would presumably only rarely give a selective advantage. Moreover, it will presumably only cause a metabolic burden to maintain this enzyme within the cell. However, in highly contaminated places or sites that are continuously exposed to PFAS, this is definitely an important consideration.
    2. The spread of the GMO is likely to occur when it is not physically restrained in, for example, a bioreactor. Therefore, the probability that the GMO eventually reaches a place where this situation might occur is high.
    3. The probability of the spread of the synthetic genes also comes down to the selective advantage of the ability to degrade PFAS. This horizontal gene transfer might occur , but will only produce a new organism with changed characteristics when it outcompetes the other biodiversity.
  4. Estimation of the risk posed by each identified characteristic of the GMO
    1. As discussed in step 3 of the ERA, the probability for a loss of biodiversity as a result of the introduction of the PFAS-degrading GMO is low. On the other hand, the consequences if this does occur would be substantial since entire ecosystems could be derailed. So taken together, the total risk is still substantial.
    2. The occurrence of the spread of PFAS-degrading GMO to a PFAS-containing site or material does not pose a large hazard. Specifically, this will only locally cause a problem and will only lead to the degradation of a certain material. However, the probability of this event is quite high , since the spread is presumably likely to eventually occur when there are no physical barriers. This consequently also increases the risk of this occurrence.
    3. As a result of the transfer of the synthetic genes, other organisms could receive these characteristics and thereby potentially pose the same threats as the GMO itself. It was also concluded that the probability of this event is similar to the possibility of obtaining a selective advantage. Therefore, both the hazards and the probability are high, and thus pose a high risk of transferring the synthetic genes.
  5. Application of management strategies for risks from the deliberate release or marketing of GMOs
    1. No management strategies are (yet) in place
  6. Determination of the overall risk of the GMOs
    1. Overall the risks are too high without a sound biocontainment strategy

Following the steps of the environmental risk assessment, we found that the possible risks associated with the GMO include undesired activity and losses of biodiversity. Taking both the potential hazards and probabilities into account, we have concluded that the overall risk of the release of this GMO, without the use of additional management strategies, is too large.

Implementation of DOPL LOCK

In order to limit these assessed risks, DOPL LOCK is implemented on the PFAS-degrading GMO. This means that the gene of interest will be cloned inside the double plasmid system, as suggested in implementation. This would lead to the introduction of a region-specific kill switch and the prevention of horizontal gene transfer through the TA systems.

The intended application for this GMO would be to remove excessive PFAS in a tank within wastewater treatment plants. Considering the environment of the application of the PFAS-degrading GMO, the inducer can not be a naturally occurring compound. This originates from the fact that the presence of this substance outside of the PFAS-degradation site, will lead to the survival and proliferation of the organism outside of the specified boundaries. Secondly, the inducer should not be degraded or decrease in concentration over the course of the wastewater treatment. This would lead to varying concentrations of the inducer during the bioremediation and thereby also affect the lethality of the GMO.

Based on these considerations, the inducible promoter pDawn was chosen to regulate the expression of the antitoxin in the final DOPL LOCK system. As a consequence, the antitoxin will only be expressed in the absence of blue-light (360 nm) [14]. This condition of darkness should be imposed on the tank by operating this facility inside. Since the presence of blue light represses the expression of the antitoxin, survival of the cell outside of the reactor is prevented. Another advantage of using this promoter is that costs that are associated with the use of chemical inducers are avoided.

Provided solution

As a result, we propose a solution to contain a PFAS-degrading GMO within a wastewater treatment plant. These wastewater treatment plants operate by continuously purifying water influent from waste streams, often with the use of microorganisms [15]. The application of the GMO could help to remove PFAS in wastewater and consequently prevent further environmental pollution. Additionally, the identified risks from the ERA are mitigated as a result of the application of DOPL LOCK. Consequently, steps 5 and 6 of the ERA mentioned above can be changed to:

5. Application of management strategies for risks from the deliberate release or marketing of GMOs
The use of DOPL LOCK as a biocontainment strategy.

6. Determination of the overall risk of the GMOs
The GMOs are now restricted to the wastewater treatment plant and are unable to transfer their genetic material. This thereby solves the problem of the escape of the organism or the genetic material. For this reason, the risk of potential hazards is decreased to an acceptable level.

Food production case-study

The United Nations (UN) have set the goal to end world hunger and malnutrition in all its forms by 2030 [16]. However, in recent years we have drifted from this goal, as the number of people who suffer from hunger has slowly been rising again since 2015 [17]. To ensure food security for the large number of people in the world, we need to increase agricultural productivity [17]. One major limitation to the yield of rice crops is stem borers. Specifically, Paddy stem borers are considered to be one of the most destructive insect pests for agricultural crops. The stem borers induced yearly yield loss has been assessed to range from 2-95% in Bangladesh, India, Malaysia and Indonesia [18]. Another big influence on agricultural yield is periods of drought, which are only expected to increase due to climate change. It was estimated that droughts significantly reduce cereal crop productionwith 10.1%, including maize, wheat, rice, barley, rye, millet and others [19]. Together, these two factors are tremendously lowering yields of important crops, thus there is a clear need for a good solution to these problems.

The 2021 iGEM team MIT_MAHE is working on a solution for these two highly impactful problems in the agricultural sector. Their solution is based on genetically modified bacteria that can live in close proximity to the plant, which ensures the necessary synthetic proteins can influence the plant without making a genetically modified plant. Their GMO will produce a toxin that can kill off the stem borers and a protein that can interrupt the normal stress response of plants to drought periods. This growth-promoting GMO will therefore make the plants resistant to stem borers and periods of drought. However, as these crops will be used as food for humans and/or animals the biosafety aspects are highly important. These bacteria should not be allowed to spread to humans and/or animals eating the food or to other plants growing nearby. So, the GMO should be contained to the controlled soil where the crops grow. DOPL LOCK could provide the solution needed to ensure these needed safety aspects.

Risks

To state the problem and to assess the sufficiency of our system, we first identified the risks that a stem borer and drought-resistant GMO would entail without the application of DOPL LOCK. We based this risk identification on the environmental risk assessment (ERA), as described in annex II of Directive 2001/18/EC and our own regulatory roadmap. This ERA also forms the basis for the license application for the approval of the non-contained use of GMOs (see Human Practices page for further details).

The steps in the ERA include:

  1. Identification of characteristics that may cause adverse effects
    1. The growth-promoting GMO will contain genes that can both disrupt the normal function of the stem borer and the normal drought stress response of the plant, thereby making the plant stem borer and drought resistant.
  2. Evaluation of the potential consequences of adverse effects
    1. The developed stem borer and drought-resistant GMO confers a selective advantage and could consequently ensure that the plants with the growth-promoting GMO will outcompete natural plants. This would lead to the gradual disappearance of plants without the capability to host the growth promoting GMO and thus a loss of biodiversity.
    2. The plants could become dependent on this growth promoting GMO to survive , since in theory all plants can host the growth promoting GMO.
    3. The synthetic genes could be spread to other wildtype organisms. These organisms thereby receive the new characteristics, in which case the spread and consequences become more unpredictable. The transfer of this synthetic material could occur through horizontal gene transfer.
    4. If the spread of the GMO gets out of hand, then all plants could become resistant to stem borers and they could go extinct. This would also result in a loss of biodiversity.
  3. Evaluation of the likelihood of the occurrence of each identified adverse effect
    1. Loss of biodiversity could occur if the growth promoting GMO is not contained to the specific place , since the resistance to stem borers and drought is a big advantage for plants.
    2. The risk of creating an overall dependency on these growth-promoting bacteria is substantial.
    3. The probability of the spread of the synthetic genes comes down to the selective advantage of the ability to be resistant to stem borers and drought. While this is highly advantageous to the plant, it is not advantageous to the bacterium. Horizontal gene transfer might therefore occur, but will not be strongly selected for by the bacterium.
  4. Estimation of the risk posed by each identified characteristic of the GMO
    1. As discussed in step 3 of the ERA, the probability for a loss of biodiversity as a result of the introduction of the growth-promoting GMO is low. On the other hand, the consequence of this would be very substantial , since entire ecosystems could be derailed. So taken together, the total risk is still substantial.
    2. Similarly, an induced dependency of plants on the growth-promoting GMO to be fit enough to survive in the ecosystem is quite derailing. Therefore, the risk of this is high.
    3. Lastly, the risk posed by horizontal gene transfer is low, as the bacteria do not gain a selective advantage by taking up the synthetic elements. The plants are the one that will gain advantage. Since the bacteria are not selected by the synthetic material, the total amount of horizontal gene transfer that will happen is probably low.
  5. Application of management strategies for risks from the deliberate release or marketing of GMOs
    1. -
  6. Determination of the overall risk of the GMOs
    1. With no added safety strategy the risks involved with this GMO are too large to get approved. The GMO could derail the natural ecosystem by creating a dependency for plants to have this specific bacteria.

Following the steps of the environmental risk assessment, we have concluded that the possible risks associated with the growth-promoting GMO include losses of biodiversity and inducing a dependency on having this GMO. Taking both the potential hazards and probabilities into account, we have concluded that the risks are too high without any added safety mechanisms.

Implementation of DOPL LOCK

In order to limit these assessed risks, DOPL LOCK would be implemented on the GMO. Specifically, this means that the gene of interest will be cloned inside the double plasmid system, as suggested in the Description page. This would lead to the introduction of a region-specific kill switch and the prevention of horizontal gene transfer through the TA systems.

Considering the environment of the agricultural plant, the inducer of the antitoxin for the region-specific kill switch can firstly not be a toxic chemical that will need to be sprayed on the plants, since this will make the food unsafe to eat. Therefore, the pBAD promoter of the proof-of-concept cannot be used, since it depends on arabinose for its expression, which can be toxic to plants [20]. A similar problem arises with IPTG-inducible promoters. Additionally, since the GMO should survive both in the paddy and within the stem borer, pH and temperature inducible promoters are also not useful.

Options for the inducible promoters are either induced with a non-toxic compound or not with a compound at all. However, due to the difficult environment settings of this GMO a non-compound activated inducible promoter is unfortunately not possible. A good alternative is the theophylline-activated P43 promoter, which is designed for use in Bacillus subtilis [21]. Theophylline, which is structurally and pharmacologically similar to caffeine, is a cheap and nontoxic chemical, enabling it to be a potential inducer for this specific GMO [22]. Furthermore, Theophylline should not naturally occur in other nearby soil, which prevents the spread of the GMO from the paddy to other naturally occurring plants [23].

Provided solution

For the agricultural site, we envision an area where rice plants are grown and treated with these GMOs. This would ensure that the yield of the plants is not reduced by stem borers and drought and thereby provide a part of the solution for the difficulties with food shortage in the world. Furthermore, the application of DOPL LOCK ensures the biocontainment of the GMOs since they are unable to leave the boundaries of the agricultural site and cannot transfer their genetic material to naturally occurring bacteria outside of these boundaries.

Therefore, steps 5 and 6 of the ERA mentioned above can be changed to:

5. Application of management strategies for risks from the deliberate release or marketing of GMOs
The use of DOPL LOCK as a biocontainment strategy.

6. Determination of the overall risk of the GMOs
The GMOs are now restricted to the bioremediation site and are unable to transfer their genetic material,thereby solving the problem of the escape of the organism or the genetic material. For this reason, the risk of potential hazards is decreased.

References

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