This page will discuss the considerations and design choices concerning the safety of DOPL LOCK. We will explore several scenarios of potential safety problems our system could face and how DOPL LOCK is designed to overcome these possible concerns. These problems are not solely about how DOPL LOCK could impact the environment or the health of people, but also about the security and usability of the system itself. Below, you can find a table outlining our main safety and usability concerns of DOPL LOCK.

Executive summary

Risk Safe-by-Design
Mutations in DOPL LOCK
Environmental concerns
Horizontal gene transfer
Physical escape
Competition wild microbes
Human consumption

Mutations in DOPL LOCK


As in all other genetically engineered platforms, mutations can occur in our system. This will lead to different possible outcomes according to the position and type of mutation. For the vast majority of these mutations, problems would not arise for the functionality of the system as a whole since we designed our system to be Safe-by-Design. Our system is inherently resistant to various types of mutations that could be a major issue to other biocontainment systems [1] due to our extra layer of redundancy by using double toxins and antitoxins.


A mutation in the toxin gene can cause toxin inactivation or a decrease its activity [2]. This would allow the plasmids to be transferred to other organisms without the effect of the toxin, thus increasing the chance of horizontal gene transfer (HGT). To minimize this risk, we have included a toxin gene on both of the plasmids. In this case, even if a mutation occurs in one of the plasmids, the other plasmid still has the toxin. This should lower the chances of transfer of the plasmids.

We have considered what we could do to prevent issues stemming from this problem. To this end, we consulted Akos Nyerges. In this interview, we received the advice to incorporate multiple toxin/antitoxin cassettes in our final design to reduce the chance of system failure as a result of mutations in the toxin genes. Another piece of advice we received was to make use of an Escherichia coli strain that does not contain transferable elements. This reduces the likelihood of mutations occuring due to the insertion of transposable genetic elements inside our safety system, increasing the overall safety of the system. Further, it is also less likely to incorporate transposable genetic elements in the coding sequences of the cargo genes on the plasmids, which would reduce the efficacy of the system.

Lastly, due to the copy-number of our plasmids, there is yet another layer of redundancy protecting DOPL LOCK from mutations. Because we chose plasmids with origins of replication (Ori's) that result in a relatively high copy-number, the chances that mutations result in HGT or the loss of a plasmid decreases. However, it should be noted that there can never be a method to fully prevent mutations in all toxin copies on a plasmid, because mutations can happen spontaneously [3]. This is a weakness inherent to DNA based systems, although the risk of this causing a problem to the use of the system can be decreased by taking these precautions.


Next to the toxin, mutations can also occur in the antitoxin genes, causing loss of function or decreases in activity. As the antitoxin is needed for the survival of the host, inactivation of this gene will kill the bacteria and the functionality of the system will be lost. We expect, therefore, that there is a selection pressure for the maintenance of intact antitoxin genes. This should greatly decrease the scale of this problem to the system.

We have identified one scenario in which this could be a problem: should one of the antitoxins gain a mutation, it would mean that there is no more evolutionary pressure for the cell to retain the plasmid. If there is a subsequent mutation on the toxin on the other plasmid, it could give rise to the possibility for the cell to lose one plasmid without induction of cell-death. However, we hypothesize that the odds of this happening is so low that it is an acceptable risk, although this needs to be proven experimentally.

Promoter sequence

Similar to mutations occurring in the toxin or antitoxin genes in DOPL LOCK, mutations in the promoter sequences should generally not be a large problem for our system. However, it is likely that mutations in the promoter sequence occur more often than mutations in the coding regions. This is because it has been shown that toxin/antitoxin systems mostly mutate in their promoter regions [4]. This makes promoter sequence mutations more likely to pose a risk for our system than mutations in the coding sequence of the genes. One way to mitigate this risk is by adding an essential gene to the toxin cassette and deleting this gene from the chromosome of the host. By doing this, strong selection pressure is created in favor of retention of the promoter sequence, since the essential gene is co-expressed with the toxin or antitoxin from the same promoter. This is a form of genetic addiction [5].

There are certain scenarios where these types of mutations could pose problems for the biosafety of our system. For example, a major concern here is that mutations in the inducible promoter could change it to act like a constitutive promoter. This would mean that our engineered kill-switch would lose its function, because the antitoxins will be constitutively expressed. This will create a plasmid that does not induce cell death when the host escapes the predefined medium or area of containment. However, we again have the safety net of redundancy: both plasmids would need to mutate their inducible promoters to become constitutive in order to allow the cell to escape. Further, there are multiple copies of each plasmid in the cell. This makes it by no means impossible for the synthetic gene to circumvent containment, but it is expected to greatly reduce the escape frequency.

Origin of Replication

Mutations in the origin of replication can have various consequences. Firstly, it could affect the binding affinity of proteins that regulate cell division and specifically, the binding of the replisome of the plasmid [6,7]. Secondly, it could decrease the binding affinity to a lower level [8]. This could result in varying copy-numbers after mutations leading to a disbalance in the molar ratios between the toxin and the antitoxin because of differential expression [9]. However, we do not regard this as a major issue. Specifically, in the case of a fully realised system, these mutations should balance themselves out due to selection pressure: when one of the plasmids suddenly has a higher copy-number, this will be a large metabolic burden to the cell [10]. In case the copy-number decreases, less of the antitoxin will be made. This will inevitably increase the lethality of the toxin on the other plasmid since the neutralisation capacity would decrease. Similarly, when the copy-number increases, there would be a disbalance between the toxin on the plasmid and the antitoxin on the other plasmid. Therefore, in both cases, the toxin would not completely be neutralised by the antitoxin, resulting in a larger metabolic burden on the cell.

Environmental consequences


In a potential GMO escape they can potentially cause ecologic harm. This can come in the form of strong competition and selection pressure against wild organisms, changes in biodiversity of soil and water ecosystems or other unforeseen consequences. These risks will differ depending on where in the environment they spread and on the synthetic DNA that is contained in DOPL LOCK. Because the toxins are unable to cross the cell membrane [11] DOPL LOCK itself is not considered as a toxic material for other organisms. We also decrease the risk to other organisms by using inducible promoters to control the GMOs' survivability.

However, it should be noted that for every application using DOPL LOCK, a risk and safety assessment must be done to ensure minimal risk to the environment, like those we did on our Implementation page.

Horizontal gene transfer of plasmids


Horizontal gene transfer can occur through many different mechanisms. For now, we will assume that the bacteria take up the plasmid through natural transformation, i.e. the bacterial cell takes up the DNA from the environment. Of course, there are other types of horizontal gene transfer, but the result of these different mechanisms remains the same regardless of how the DNA enters the cell. We assume this method of plasmid transfer because it can occur regardless of specific factors that might influence HGT. For example, conjugation requires an origin of transfer [12] on the plasmid to occur, but natural transformations do not require DNA machinery like conjugative elements or genes like recA [13]. Because origins of transfer pose a risk to the safety of our system by means of conjugation, we chose to remove it from our final plasmid designs.

Plasmids spread to a different species of bacteria

This scenario revolves around the main issue our system is designed to deal with: how to prevent the spread of plasmids containing synthetic genes between different bacteria? On our Implementation page, we discuss that our aim is to select toxins for our end-product with a range as broad as possible. One possibility of making a system without using natural antitoxins would be to use nucleases as toxins [14], with antisense RNAs (asRNAs) functioning as the antitoxin [15,16]. This would be beneficial because it would mean no intact DNA is left behind after induction of cell death.

In theory, when one of the plasmids spreads to another bacterium, our system should prevent the negative consequences that could originate from this transfer. There are several factors that all have to align for the containment to fail.

Firstly, the origin of replication of the plasmids needs to be active in the new host for the plasmid to be maintained in the cell. Secondly, the receiving bacterium needs to be resistant to the toxin on the plasmid. Thirdly, the ribosomal binding sites have to function in the new host. Fourthly, the promoter-sequence needs to be active in the new cell for the genes to actually be transcribed. The final and perhaps most critical condition, is that there should be a selection pressure in favorof plasmid retention for the microbe. If there is no selection pressure to keep the plasmid in the cell, it is likely that the plasmid will be lost, since it will come at a great metabolic cost to the new host to maintain the plasmid and express its proteins. This would make outcompeting other bacteria difficult. All in all, the chances of a plasmid spreading are considered to be quite slim.

Plasmids enter cell with native antitoxins

In the case that one plasmid enters the cell of a bacterium with a native antitoxin present, it is unfortunately possible that the cell will continue to proliferate. However, this does not mean that the system will not work anymore. The high copy number of the plasmid means that there will likely be more copies of the toxin gene, than the antitoxin in the bacterial chromosome. Therefore, as the toxins will be under constitutional promoters, there might be a disbalance between the native antitoxin and the introduced toxin. It has been shown that overexpression of toxin genes leads to growth inhibition [17,18]. Having a native antitoxin in the cell is therefore no guarantee for escape from the system, although it presumably reduces the killing potential of DOPL LOCK.

Horizontal gene transfer of both plasmids

One of the biggest hurdles for our system to overcome is when both plasmids are horizontally transferred at the same time. Currently, DOPL LOCK has not implemented a way to prevent this from happening. However, how much of a safety concern this is, remains to be answered. Provided that the system works in the way that it is designed, the bacteria will remain physically tethered to a predefined area because of the inducible promoter before the antitoxin gene [19]. The new microbes that contain the double plasmids will simply be a part of the application of the synthetic material. In addition, the possibility remains that the microbe is unable to express the synthetic genes that are added on the plasmid. This is the case whether that be due to disparities in ribosomal binding sites [20], replisomes that are unable to bind to the plasmid, wrong ratios of G/C content [21], or another reason. Having said that, this remains one of the, if not the largest risk pertaining to horizontal gene transfer inherent to our system. A solution to this problem would be to integrate one of the plasmids in the host chromosome.

Phagocytosis by microbes

Microbes directly compete with each other and in doing so, also sometimes consume each other. When they do, there is a chance that some of the plasmid DNA remains intact. In fact, it has been reported that bacterial predation increases rates of HGT dramatically [22,23]. It has also been demonstrated that at least some eukaryotic cells are able to take up DNA from other cells through phagocytosis [24]. We deem it possible, but unlikely, that the genes in DOPL LOCK could be transferred horizontally by means of phagocytosis of cells containing DOPL LOCK.

Escape of bacterium from predefined condition


Microbes are often motile organisms, being able to swim [25], pull themselves along surfaces [26] or float through the air [27]. Therefore, it is important to keep this in mind when designing our system. We consider building a physical containment structure for GMOs is a good starting point for biocontainment strategies, but it should not be the only measure, especially in semi-contained applications.The consequences of escape from the predefined areas can be grave [28]. Physical biocontainment barriers, such as bioreactors and pressurised chambers are vulnerable to terrorism and environmental disasters. Therefore, we advise that in order to lift biocontainment strategies to the next level, one requires more than solely a physical barrier for genetically modified organisms (GMOs), especially in (semi-)contained environments. Semi-controlled applications of GMOs entails applications in an environment where localised containment is possible, yet with a clear physical path for GMOs to spread into the environment. This is different from non-contained use, where GMOs are released without using any physical containment measures, such as Oxitec's mosquitoes [29].

Our system provides a means of biocontainment through the use of inducible promoters upstream of the antitoxin genes. This therefore functions as a kill switch, which decreases the survivability of the cells upon a change in the presence of inducers. In our experimental phase, we used the pBAD promoter which is induced by arabinose. In practice, the specific promoter used will depend on the application of the system. Placing the antitoxin under an inducible promoter allows for careful selection of the physical places the microbes containing our system can survive. Without the inducer present, no antitoxin will be made to counteract the toxin. Therefore, the GMO will soon die.

Recombination events


One possibility for horizontal gene transfer that is often overlooked, is (homologous) recombination. This can happen in several ways, but it means that (part of) the DOPL LOCK DNA is introduced into another sequence. This can result in varying outcomes depending on the location and identity of the recombining sequences. Therefore, it is not possible to make generalising statements on the effects of recombination. However, there are a few risks that can be identified to the functioning of DOPL LOCK as a biocontainment system with regards to recombination events. We explore these events in the scenarios below.

Homologous recombination into bacterial chromosome

In case of extensive homology with genes from the bacterial chromosome, it would presumably only be a matter of time before one of the plasmids recombines with the chromosomal DNA of the host, since 20 bp has been shown to be enough for recombination between the chromosome and plasmids in E. coli [30]. Should this happen with DOPL LOCK, there is a possibility that bacteria lose both plasmids but still carry some of the introduced DNA. Therefore, it is imperative to make sure that there is as little homology as possible between the host chromosome and the plasmids [31]. It is, however, not unthinkable that this could happen in the case of for example double horizontal gene transfer to a species with the antitoxin native to their chromosome. In such a case, the synthetic DNA could become a part of the chromosomal DNA for good. This is not an ideal situation, since this can have an impact on the environment, depending on the nature of the introduced DNA. Therefore, it is of utmost importance to design the vector to have a minimal amount of DNA that could possibly recombine with other plasmids or chromosomal DNA.


As is the case for viruses in animals, bacteriophages have had a significant influence on the genomic evolution of bacteria [32]. It is inevitable that the bacteria which harbour DOPL LOCK will be infected by bacteriophages. This could lead to recombination events that could in turn cause large scale horizontal gene transfer.

The odds are low, but nonetheless, since it has been shown that bacteriophages are major drivers of horizontal gene transfer in bacteria [33,34], there is a grave risk that synthetic DNA contained in DOPL LOCK would recombine with DNA from the bacteriophage. After this, it could end up in the chromosomal DNA of other bacteria if they are in turn infected by the same, recombinant bacteriophage. If this happens, there is also a chance that selection markers or other plasmid DNA could integrate into the phage genome and spread it to other bacteria [35].

Currently, there are no realistic strategies that could keep bacteriophages from infecting bacteria completely, even in the lab [36]. Bacteriophages and bacteria have co-evolved for many years, and virtually every defensive mechanism evolved by bacteria has been overcome by some type of bacteriophage [37]. Similarly, any system that is put into the cell to prevent bacteriophage infection will probably quickly be rendered ineffective following exposure to bacteriophages. This is due to the incredible mutation-rate of bacteriophages.

The issue remains, however, that bacteriophages are a real possibility for synthetic DNA to spread across bacterial phyla. Currently, DOPL LOCK can provide no strategy to counter this phenomenon, especially since phage DNA often remains 'silent' in bacterial chromosomes after it is inserted [38].

Recombination of both plasmids

Another concern for DOPL LOCK is the recombination of both plasmids. This would disrupt the system because if Romeo and Juliet recombine, it will allow for the spread of the entire system simultaneously. In turn, it would be more likely that the plasmid is transferred horizontally. The main strategy to prevent this from happening is by ensuring minimal homology between both plasmids. This is one of the reasons it is vital that both plasmids have a different Oris, selection markers and TA genes.

DOPL LOCK microbes are outcompeted by other microbes


There is a real concern that, by using a system that is relatively large and can offer a large metabolic burden to the cell, there will be a strong selection pressure against retention of both plasmids [39]. This could lead to microbes carrying DOPL LOCK being outcompeted by other microbes [40,41]. Consequently, it will become difficult for these bacteria to fulfill the task that they were designed to do, but this is by no means a safety concern. In fact, in use-cases like bioremediation, this may serve as a feature, rather than a problem. When DOPL LOCK containing microbes are released in contaminated soil that inhibits microbial growth, after detoxification, this could automatically create selection pressure against DOPL LOCK. This means that DOPL LOCK has the potential to remove itself from the environment after fulfilling its task: making the environment liveable for other microbial communities. Another method to achieve this same effect could be to make the toxin inducible as well, so the bacteria can be killed after they completed their task, depending on the specifics of the application of DOPL LOCK.

Applications in bioterrorism


Bioterrorism is an imminent threat associated with developments in biotechnology and genetic engineering platforms [42]. These developments will heighten the possibility of biotechnology being used in a harmful way, thus causing disease to animals or humans.

In theory, it would be possible to use DOPL LOCK in a nefarious way, by making plasmids which contain genes including an origin of transfer so that suddenly the system can be transfected to other bacteria.

However, this is not unique to DOPL LOCK and is not a risk inherent in the system itself, but in the use of GMOs in general. Even with an origin of transfer, our system will still include the toxins and conditional promoters that should tether the genes and bacteria in place. Additionally, it is possible to make a 'genetic watermark' [43] on each plasmid. This could ensure that whenever a DOPL LOCK plasmid is found in a place where it does not belong, this watermark can be sequenced. Subsequently, this watermark could be compared to a database and using sequence analysis, it would be possible to trace back which user of DOPL LOCK released these bacteria. This would not stop bioterrorism from happening, but it would make it less appealing to do so, since the traceability of the system likely disparages nefarious use of the plasmids. These databases could be integrated in the process of obtaining licences for GMOs.

Because DOPL LOCK inherently counters the unbridled spread of GMOs into different environments, the system inherently is unattractive for the purposes of bioterrorism. We therefore do not see a significant risk of bioterrorism applications of DOPL LOCK, especially when used in tandem with a personalized barcode system.

Further, we recommend any (future) platform that potentially uses GMOs into semi-contained environments to make use of these kinds of barcode systems to improve accountability and traceability. This would ensure that after the discovery of unintended release of GMOs, it will be easier to do an analysis of what went wrong to prevent future incidents. Further, subsequent coaching to said parties to improve containment measures might reduce future incidence of unintended release of GMOs.

Consumption by humans


One of the main concerns of using GMOs in different settings is the danger it can pose to humans. Generally, the risk differs in relation with the type of organism. The exposure of humans to these GMOs can happen in different ways: by being dispersed in human food, water or even indirectly by being consumed by the animals which will later be used in food production. Consuming microbes could affect the human body's natural microbiome [44]. However, we do not see any reason that DOPL LOCK should be toxic on its own. Firstly, the toxin-genes currently encoded by DOPL LOCK are already present in the human gut, since they are derived from E. coli and are not part of its virulence factors [45]. Of course, depending on the actual toxin gene implemented in the final system, a new evaluation should be made on this matter.

If this is still the case however, this means that no novel genes will be added, except for the cargo that is loaded onto the plasmids and any novel biosafety genes. It is not a large problem if these plasmids are then transferred to native E. coli, because of differential expression levels creating a negative selection pressure for DOPL LOCK.

On top of this, the toxins in DOPL LOCK are proteins and RNA, which are not able to cross cell-membranes without proteins that traffic them [11]. Therefore, microbes containing DOPL LOCK are generally not toxic to other microbes in the environment, but this can vary depending on the microbe used.

Of course, safety evaluations should be done for the cargo on a case-by-case basis with regards to toxicity to humans. The most likely risk that we can identify to human health is the possibility of DOPL LOCK outcompeting native gut microbes, creating a disbalance in the species diversity which can lead to a wide array of negative outcomes [46]. Therefore, until much more research has been done on the effects of introducing exogenous microbes and plasmids into the gut microbiome, we strongly discourage any attempted applications of DOPL LOCK for human consumption or insertion into the gut.


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GMO and Lab Safety Course

As working safely in the laboratory is considered one of the most essential aspects of safe scientific conduct, we have complied with several safety protocols. All organisms used in our project are common, non-pathogenic laboratory strains. Besides, all team members have taken GMO courses from Leiden University that give instructions on how to use GMO's in a safe way. All the rules have been reiterated on the first day of laboratory work, which, for example, consist of putting on a lab coat when entering the lab, not drinking or eating in the lab, using ethidium bromide in a safe way and being responsible in our use of GMOs .

Waste safety

For waste safety, we had special bins for GMO-contaminated waste and a separate bin for objects contaminated by ethidium bromide. These bins were disposed of according to the safety rules at Leiden University.

Safety for COVID-19 pandemic

Spending our lab time in the COVID-19 pandemic, we had several measures in addition to the fact that we all have been vaccinated. Each team member got a self-test two times a week and kept a distance of 1.5 meters in the lab. Most of our meetings were held online.

Ethical issues

There are several ethical questions that we should consider when implementing our project. Some of the questions we should answer are 'Considering all the risks mentioned above, is it still beneficial to use our system?' and 'Should we even strive to create a world where we release more GMOs?' Even though our system has significantly reduced the risks, there is always the possibility of making mistakes.

In our Human Practices page, we have discussed with experts and also the public to understand their perspective about these issues. We also use a survey to better understand the opinion of people on using GMOs. We wrote more on this on our Entrepreneurship page.