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.
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.
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.
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]:
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.
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.
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.
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.
The Detection tool itself does not involve any synthetic biology applications
and does therefore
not pose any of the threats described above.
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.
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 (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].
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.
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.
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.
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.
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.
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.
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.
Biosafety
Biocontainment
General Risk Assessment
Biosafety Risk Assessment
Detection
Delivery
Remediation
Spread
