Team:Wageningen UR/Safety/Biosafety



Synthetic Biology can be explained as the design and construction of a new biological system or the re-design of an existing system for human purposes. Another term used instead of Synthetic Biology is Intentional Biology, further highlighting the engineering approach [1]. However, the designed system can behave in ways different than intended and cause safety risks for the environment and society. These concerns are addressed under the term of biosafety, which describes “the containment, principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release” [2]. It is within a researcher’s responsibility to implicate social, ethical and legal aspects, balance between proaction and precaution, and mitigate negative impact of new technologies [3]. To do so, risk assessments are being conducted to evaluate the potential negative impact research could cause. The risk assessment of the four pillars of Xylencer can be found below.


The use of GMOs in biotechnological applications is already widely accepted and applied in industry. These GMOs are highly contained in this setting though. There is almost no chance of release.

As responsible scientists, we believe that there is a need to control GMO work as it is difficult to foresee the consequences of accidental and intentional release. This control promptly diminishes when a GMO is released into nature, resulting in a lack of containment of the organism. Outside the laboratory, you will not have the same control over a GMO as in a highly contained laboratory setting where the strain is adapted to laboratory standards. When a GMO is introduced into nature, it must endure competition and other types of abiotic and biotic stresses from the environment. Evolution drives the organism to change in order to adapt to these new circumstances. It is not possible to completely control or predict these processes.

Currently, multiple strategies are developed which are intended to control GMOs with unintended (escape from the lab) or intended (introducing a GMO in nature) release. There are three mechanisms that we wanted to highlight that are currently used as biocontrol mechanisms, namely auxotrophy, recoding and kill switches. Xylencer uses a kill switch for biocontainment.

Kill switches are systems inducing lethality in a GMO once it leaves the laboratory setting. The most famous kill switches are the “Deadman” and “Passcode” systems [7]. These systems can be altered to fit the desired molecules of choice. The “Deadman” system uses a continuous stimulus from the environment to repress a toxin. When this stimulus disappears, the repression is removed, so the strain will switch to toxin production. The “Passcode” system works differently. It uses transcriptional repression, but multiple stimuli are needed to repress the toxin. Another stimulus needs to be absent. If these requirements are not all met, the toxin is expressed, and cell death will occur.

Kill switch: A kill switch based on a toxin/antitoxin system. When the toxin is present in higher concentrations than the antitoxin, the bacterial cell is killed.

Xylencer also includes a kill switch for the Phage Carrier Bacterium (PDB). The PDB requires a kill switch that is based upon time. This will allow the chassis to be able to survive for a determined time in the field and allow it to detect X. fastidiosa and produce phages. The PDB kill switch relies on the previously mentioned “Deadman” and “Passcode” system principles, although now we use time as our toxin accumulation system. The PDB kill switch is based on the circadian oscillator system from Synechococcus elongatus PCC 7942 [8]. This system will be controlling the MazE (antitoxin)/MazF (toxin) system, a natural system that is present in many different species, including E. coli. The underlying principle is that the oscillator is able to increase the amount of MazF over-time, where MazE is constitutively expressed. Ultimately, the MazF concentration will exceed the MazE concentration, resulting in cell death [9].

In our case, the oscillatory kill switch is a backup system. If our carrier bacterium encounters Xylella fastidiosa, it will induce phage production. Phages are able to lyse the PDB cells, which results in phage mediated cell lysis and death of the bacteria.

In general, each kill switch that relies on a timer would be suitable for our carrier bacterium. The TU Eindhoven 2014 iGEM team proposed (although did not test) a system that is based on pulses increasing in strength with every pulse [10]. Coupling this to, for instance, the MazE/MazF system, could be a suitable option for our carrier bacterium.

Synthetic auxotrophy relies on the addition of a molecule to the growth medium in laboratory settings that is required for the growth of your organism. This specific molecule is not present in nature and the strain cannot synthesize this molecule itself. As a result, if this engineered strain escapes the lab, it will not be able to survive in the environment. These strains are often created by mutating existing strains. It is often relatively easy to evolve around auxotrophy.

Auxotrophy: A specific molecule (blue star) is needed for the growth of the bacteria. The lack of presence of this molecule leads to bacterial cell death.

The principle of the technique of recoding is that, for instance in the case of biocontainment, all known UAG stop codons will be replaced with UAA codons [4]. This allows the former stop codon (UAG) to be used in this strain as a new codon, coding for a non-standard amino acid (NSAA) [5,6]. Scientists redesign essential enzymes to depend on this unnatural amino acid. This dependence means that the organisms will not survive outside of the lab as the translation of their essential genes into enzymes is hampered. This can be defined as a synthetic auxotroph. Bacteria are less likely to evolve to circumvent the biocontainment.

Recoding: recoding can create a synthetic auxotroph.

General Risk Assessment

According to the Cartagena Protocol on Biosafety to the Convention of Biological Diversity (CBD), risk assessment means to “identify and evaluate the possible adverse effects of living modified organisms” [11]. Traditionally, risk assessments involve the identification of hazards and the estimation of exposure to the environment or society and provide supporting information for policymakers. Further, the CBD states “[...] all risk assessments of living modified organisms should be conducted on a case-by-case basis as the impacts depend upon the trait inserted, the recipient organism, and the environment into which it is released” [11]. Whereas this approach of risk assessment is suitable for GMOs, synthetic biology poses new challenges to hazard identification and exposure estimation. Synthetic biology includes the introduction of various biological parts or pathways into living organisms, or even fully synthetic cells, as compared to single genetic changes in classical biotechnology procedures. The interaction of various parts and traits and the lack of comparable natural counterparts result in rising uncertainty levels, causing classical risk assessment to reach its limits [12]. Challenges of synthetic biology in risk assessment [12,13]:

  • Predictability arrow_downward

    The manipulation of metabolic pathways or their substitution with synthetic pathways and gene circuits lower the predictability of how a system will behave. In addition, the amount of possible interactions rises with the number of parts added to an organism, greatly increasing the uncertainty of a synthetic system. These interactions could result in the production of toxins, virulence factors or other harmful substances. They can make changes in the lifestyle of the organism.

  • Evolutionary Forces arrow_downward

    Through evolution, parts can be altered, deleted or even multiplied and therefore, influence the network and its functionality. These changes are difficult to predict, which results in the altered parts and their new interactions being impossible to foretell.

  • Robustness & Reliability arrow_downward

    Robust gene circuits that are less prone to alterations and assessed for reliable functionality decrease uncertainty and the influence of evolutionary forces on the networks. Still, guidelines are missing defining on how to test for robustness & reliability and in which unit to express it. These guidelines might vary depending on whether they are applied in e.g. a contained environment or used to perform medical treatment on humans.

  • Hazard arrow_downward

    Synthetic organisms could pose a threat to society and environment if being released from their contained environment. However, some synthetic biology applications are meant to be used and are functional outside a contained environment in specific habitats. By adopting and spreading to new ecological niches, synthetic organisms can interfere with existing ecosystems. Furthermore, microorganisms are known to exchange genetic material efficiently between each other, which could be hazardous to human health and environment in cases such as antibiotic resistance genes or toxins.

Biosafety Risk Assessment


Detection Button

The Detection tool itself does not involve any synthetic biology applications and does therefore not pose any of the threats described above.


Delivery Button

Phage therapy is a very promising technique for combatting bacterial infections, but application in agriculture suffers from problems in the delivery of phage particles. In  the second pillar  of  Xylencer,  we create a Phage Delivery Bacterium (PDB) to overcome these issues. 

  • Choice of Chassis arrow_downward

    Chassis organisms should be non-pathogenic in order to reduce potential hazards and to regain some degree of predictability. To reduce the risk associated with using Xanthomonas as our chassis, of which some species are known plant pathogens, we have performed extensive literature research, bioinformatical analysis and applied machine learning to find a genetic basis for non-pathogenicity. Via this procedure, we are able to predict non-pathogenic Xanthomonas species fitting the needs of our PDB. Furthermore, Xanthomonas is a bacterium already extensively used in the field of biotechnology as it produces the valuable polysaccharide, xanthan [14]. Considering the pathogenic origin of various other chassis, such as E. coli and P. putida, the establishment of Xanthomonas as a future chassis has potential, especially as its genome is sequenced and pathogenicity related genes are identified [15].

  • Horizontal Gene Transfer arrow_downward

    Horizontal Gene Transfer (HGT) plays a major role in Xylencer. Our chassis will be placed outside a contained environment and will interact with X. fastidiosa, which is known to be naturally competent for gene transfer, and other microorganisms [16]. Henceforth, an analysis of the chassis’ original genes, the inserted genes, their interactions, and their effects on the environment and the pathogen’s virulence has to be conducted. Neither the gene circuit consisting of the inactive Cas9 (dCas9) protein and the AntiCRISPR (Acr), nor the Diffuse Signaling Factor (DSF) receptor complex are involved in toxin production. The CRISPR-Cas system's original function is immune defense in bacteria against phages [17]. The transfer and adaption of the catalytically dead version of the Cas9 gene into the X. fastidiosa genome could, therefore, result in the pathogen to be resistant against phage therapy. Regarding the Acr no risk is to be expected. These proteins are the counter mechanisms of phages against the immune system of bacteria, the CRISPR-Cas system [18]. Acquiring Acr’s would rather pose a disadvantage as it would turn off the phage direct immune defense of a bacterium, in case it possesses a CRISPR-Cas system. Regarding the DSF receptor complex, it is either directly derived from X. fastidiosa or consists of homologs from the closely related Xanthomonas species, resulting in the risk to be considered low. However, DSF perception is linked to transcriptional alterations and the effect of copy number variations or overexpression on the system is hard to foretell [19].

  • Biocontainment arrow_downward

    Besides assessing the risk the transfer of specific genes could cause, measures can be taken to reduce the overall chance of HGT to take place. Interesting examples are the “Gene Guard” of the Imperial College iGEM team 2011 and the “bWARE” system of the Paris Bettencourt 2012 iGEM team . We have personally not incorporated these systems in our chassis. Furthermore, the avoidance of mobilization elements needed for conjugation or transduction reduces HGT [20]. A constant point of concern is also the spread of antibiotic resistance genes as incidents with multi drug-resistant bacteria occur more often. Auxotrophic markers represent a sound solution as mutations to overcome auxotrophy are rare and no transgenes are added to the system [21]. Next to auxotrophic markers, induced lethality, more commonly termed “kill switch”, can be used to prevent the survival of our chassis in the environment and therefore reduce HGT. The toxin used in our kill switch could be linked to the installed gene circuit, while the antitoxin would be placed in a more distant part of the genome or in a different plasmid. This way, in the case of HGT of our gene circuit to another bacteria, the toxin would be transferred, but not the antitoxin, leading to cell death of the recipient bacteria.


Remediation Button

Although  extremely promising, there are some difficulties  associated  with the  use of phage therapy  on its own. To improve phage therapy, we form an alliance with the plant by artificially triggering  a  systemic  plant immune response.

  • HGT of MAMPS Genes arrow_downward

    Bacteriophages are known to act as vectors for the transfer of genetic material between bacteria via the process of transduction [22]. We therefore assessed the risk associated to a situation in which X. fastidiosa or other microorganisms take up and insert the flg22 coding sequence into their genome and found it to be negligible. flg22 represents the highly conserved amino acids (aa) 30 – 51 of the in total 394 aa long FlaC protein of Pseudomonas aeruginosa [23]. FlaC does not possess toxic characteristics and we assume that flg22 does not represent a functional variant of the fully sized FlaC protein as it is much shorter. Besides, flagella are a conserved cell structure naturally present in many bacteria. If the transfer of the flagella gene cluster to other bacteria poses a threat, this threat would already be present without the Xylencer phage. In fact, expressing flg22 would be deleterious to the fitness of X. fastidiosa’s or any other plant pathogen, as the peptide acts as an elicitor of plant defense responses.

  • Host Specificity of Phages arrow_downward

    Compared to viruses affecting humans, animals, and plants; bacteriophages exclusively infect bacteria. This is due to phage infections relying on very particular molecular interactions between the phage and their bacterial hosts. These phage-bacteria interactions are so specific that “the record of phages crossing generic boundaries is small when compared to the very considerable number of genus-specific phages” [24]. The lytic phages of X. fastidiosa have been found to be lytic for various strains of the pathogen, collected from different host plants, and few of its close relatives of the Xanthomonas genus [25,26]. The phages’ host range apart from specific Xylella and Xanthomonas strains is still to be determined completely as they have been discovered quite recently. Further experiments have to be conducted to test for the effect of these phages on the microbiome of X. fastidiosa infected plants. Even though, a scenario in which the phages of X. fastidiosa affect other (plant- or human-related) bacterial species cannot be excluded with certainty, the chance for this scenario to occur is considerably low. For instance, the use of bacteriophages in the food industry is approved by the Food and Drug Authority (FDA), USA, under the classification of “generally considered as safe” (GRAS). We spoke with an expert in the field of phage plant microbiome interactions, Professor Britt Koskella, UC Berkeley, who stated that the effect of genus-specific phages on the entire microbiome of plants is negligible.


Spread Button

Wild plants carrying X. fastidiosa but not showing symptoms often act as disease reservoirs for (re)infection of nearby fields. To eradicate these pathogen reservoirs, we equip the Xylencer phages with the ability to spread making use of the same spreading mechanism the pathogen applies.

  • HGT of Adhesion Proteins arrow_downward

    As mentioned under the pillar Remediation, bacteriophages are known to transfer genetic material via transduction [22]. As for the flg22, we also assessed the risk associated to the fusion of adhesion proteins to the capsid of the Xylencer phages. In Xylencer, we are experimenting with a range of X. fastidiosa derived and underived adhesion proteins posing different risks. In general, the adhesion proteins are fused to the C-terminus of the phage’s capsids, meaning that their start codon ATG was removed. This way these proteins cannot be expressed without the phage’s capsid protein fused to the N-terminus. In addition, specific domains guiding the adhesion proteins to the cell membrane and integrating it are missing. This way, the chance that a bacterium displays these adhesion proteins on its cell surface, and by that, gaining surface adherence ability, are reduced. However, besides the chitin binding ability, the adhesion proteins may possess further functions, as is true for the X. fastidiosa derived protein domain HxfAD1-3. In X. fastidiosa, HxfA is known to play a role in biofilm formation and colonization of the insect vector [27]. Please see our Integrated Human Practices (Rodrigo Almeida) for more information.

  • GMO Release & Spread arrow_downward

    Xylencer’s hypothesis is that the release of the PDB in nature can be well controlled by the kill switch mechanism and the phage mediated lysis. However, the Xylencer phage is a genetically modified entity, able to spread itself via an insect vector. Even though, the spread of the phage is part of the idea behind Xylencer, the spread should have its limits. Previously, there has been shown that the capsid fusion of phage Lambda with GFP reduces the assembly and infection efficiency of phage Lambda [28]. The Xylencer phage with an adhesion protein fused to the capsid protein might therefore also experience a negative impact on their fitness. The hypothesis is that the phage, due to reduced fitness and evolutionary pressure, will lose the gene fusion after a few generations. Furthermore, Xylencer consists of different modular parts. If the spread of the engineered phages does not turn out to be limited, the PDB itself can be engineered to produce the capsid protein fusion. This way the phage itself is not engineered even though it carries the adhesion protein. However, the phage will lose the adhesion protein and therefore its spreading ability after one generation.

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