General lab safety
Fortunately, our university allowed us to enter the lab this year from June to October despite the COVID-19 pandemic. TU Delft actively kept track of the news and followed instructions from health authorities, the National Institute for Public Health and the Environment (RIVM). We followed these instructions carefully.
- The team followed strict safety measures to prevent spreading of the COVID-19 virus. We were vigilant with maintaining personal hygiene by frequently and properly washing our hands, coughing and sneezing into our elbow, and using paper handkerchiefs.
- In accordance with the RIVM (Dutch National Institute for Public Health and the Environment) guidelines, we did not go to university if we had COVID-19 related symptoms or if we had been in contact with someone who tested positive.
- The TU Delft has taken several additional cleaning measures, including but not limited to, frequent cleaning of workplaces, toilets, meeting rooms and coffee machines. They also provided us with cleaning supplies such as 70% Ethanol and hand gel to make our work as safe as possible.
- In order to keep the occupancy of our buildings and laboratories as low as possible, all student projects have been carried out off-campus. We were permitted to work in the lab with a maximum of 4 people, maintaining a distance of 1.5m at all times.
- During the summer months, we were allowed to use a large lecture hall as an ‘office’, although working from home remained the norm. Throughout the project we always made a serious effort to keep a safe distance, while at the same time trying to do our work in the best possible way
The wetlab part of our project was performed in the ML-1 area, which is situated at the Bionanoscience department of our Faculty of Applied Sciences on the Delft University of Technology campus. ML-1 is considered to be the lowest biosafety level, corresponding to BSL-1. After passing the mandatory ML1 tests we were allowed to enter and work at two labs.We worked in one regular ML-1 lab and one ML-1 lab meant for bacteriophage work only (Figure 1).
Prior to starting our experiments, all (wetlab) team members passed the obligatory safety tests about:
- General safety of the Faculty of Applied Sciences
- General Laboratory safety
- Biological safety ML-1
- Laser safety for Laser Users
Below you can find a summary of the rules employees and students at the TU Delft have to follow to guarantee a safe work environment:
In case of emergency
- In case of emergency or a minor accident we must call the internal alarm number. We must explain who we are, where we are and what the emergency is. The emergency centre of TU Delft will send the in-house emergency response team (Bedrijfshulpverlening, BHV). If needed, the emergency centre of TU Delft will contact the external emergency services.
- In case that the alarm goes off we should first think of our own safety. We must secure our workstation and leave the building as soon as possible, taking the nearest (emergency) exit. If possible, we must warn colleagues and assist people with disabilities. We have to go to the nearest assembly point/checkpoint. At the assembly point/checkpoint, we have to wait for instructions of the in-house emergency response team (Bedrijfshulpverlening, BHV).
- When we see gas, smoke or fire we must first think of our own safety. We have to press the red fire alarm button. If we feel confident we can extinguish small fires. If possible, we must warn colleagues and assist people with disabilities. We have to go to the nearest assembly point/checkpoint. At the assembly point/checkpoint, wait for instructions of the in-house emergency response team (Bedrijfshulpverlening, BHV).
- We must always report incidents, accidents, near misses and unsafe situations to the Health, Safety and Environmental advisor of the faculty.
ML-1 Laboratory safety guidelines
Waste disposal guidelines
In addition to completing the safety tests, we also received lab training on how to safely operate most of the techniques used in basic experiments. We agreed upon rules on how to work safely in our lab to minimize potential risks to laboratory personnel and the environment. We also learned how to discard different types of waste and how to minimize contamination risk. To work in the special bacteriophage lab we received a separate training, as extra safety measures are required to contain the phages and prevent contamination. We learned how to clean phage contaminated surfaces properly and how to correctly perform experiments with phages.
Apart from working safely we also made sure we designed experiments that we could perform safely. We wrote a safety report to evaluate the risks of our experiments including: experimental details, designs, safety regarding COVID-19 and biological safety information. This safety report was approved by Susanne Hage, wetlab coordinator of the department of Bionanoscience of the TU Delft, and Marinka Almering, Biosafety Officer of the Faculty of Applied Sciences of TU Delft. When we made adjustments to our project, we updated the proposal and got it approved before starting the new experiments.
Safe Experimental Design
We designed all our experiments to comply with the ML-1 safety level. Classification of microorganisms or toxins can differ between countries or regions, we followed the legislation of the TU Delft. Prior to working in the lab we checked that all organisms, vectors and parts are in accordance with the ML-1 level and the Dutch law.
To comply with our ML-1 lab we made sure that all our microorganisms were ML-1. We used E. coli BL21 (DE3) and E. coli DH5 alpha as our chassis organisms. In addition we used E. coli BL21 (DE3) as a host for our phage engineering experiments. We decided to work with the T7 bacteriophage, as it is also classified as ML-1. We exclusively performed phage experiments in a separate bacteriophage lab to prevent unintended E. coli infection (Figure 1).
We made sure to express safe inserts. The Cry7Ca1 toxin is specific to insects, and can be used in an ML-1 lab without separate acceptance from the GMO bureau.
We also expressed YmdB (RNAse II inhibitor), Mini-III (cleaves shRNA), 4-hydroxybenzoate Decarboxylase (degrades phenol) (for more information see Design). These molecules were all not toxic and classified as ML-1. All of these molecules have not been reported to be harmful to humans.
For our research we used different backbones vectors: pKD46, G322 - pUC57 - OriLR - deGFP, pTWIST_bsdBCCD_CO, pSB1C3_BPUL_GA, pTWIST_Cry, pBbB7a - GFP, pBbA2k - RFP, pBbE8c - RFP, pCas9-CR4, pKDsgRNA-p15. Some of these plasmids were kindly provided by other labs from the BN department and some were ordered via Addgene. Material transfer agreement (MTA) regulations were followed according to the rules.
We used several chemicals that are hazardous, these include Ethidium bromide, 2X Laemmli Sample Buffer and SYBR™ Safe. We have only worked with these chemicals in low concentrations and solely worked with these toxins in the designated area. This area was marked with special tape, and we agreed to all work with gloves in that area; these gloves were colored differently compared with the general stock. In addition, any contaminated equipment did not leave this area.
Safety is one of our important design requirements. PHOCUS is a targeted bacteriophage-based biopesticide used to control the desert locust crisis. Our aim is to kill the locusts by producing toxic molecules in their gut. These molecules will be produced by the gut bacteria after infection with our engineered bacteriophage. The toxins we want to produce are the locust specific Cry7Ca1 toxin and short-hairpin RNAs (shRNAs) that target essential locust genes (for more information see Design).
Whilst working on our project, we learned that in biology, nothing is 100% safe. Therefore, we decided to focus on identifying potential risks and having appropriate risk management measures in place to reduce these to an acceptable degree (Figure 1).
We make use of phages for toxin delivery. These are viruses that can only infect bacteria . An identified safety concern is that our engineered bacteriophages could be dangerous to humans. However, we learned that this risk is negligible because:
- Phages are not able to infect human cells. Even though they can enter the human body, allergic reactions are not common in humans [2, 3, S. Hagens, personal interview].
- Phage stability in certain conditions varies for different phages . For our project, it is important to choose a phage that is stable at the pH of the desert locust gut, which is pH 7-8 [J. Vanden Broeck, personal interview]. A phage that is stable at this pH is not likely to survive at the pH of the human stomach (pH 1-2) .
- If phages do survive at human stomach pH, they are not likely to affect the human gut microbiota [S. Hagens, personal interview]. Nonetheless, more research on the exact effect of lytic phages on the gut microbiota has to be done [6,7].
An additional safety concern is that our phages could be dangerous to animals and/or insects. Research has looked at the influence of phages on animal microbiota, but this impact is not well understood yet . Regarding insects, research has been done on the effect of oral administration of phages by flies and bees [9, 10]. This seemed to have little effect. All in all, more research regarding the effect phages have on the microbiota of animals and insects should be done (see Field trials).
Another safety concern is the persistence of our engineered phages in nature after being released. However, the chance of our phages persisting for a long time is low as:
- Phages are unstable when exposed to persistent levels of UV [J.B. Jones, personal interview, 11]. Thus, outside the locust gut, phages will not persist long.
- Phage stability varies with temperature. The risk of persistence in nature may be reduced by choosing a phage that is predominantly stable at locust body temperatures; 25-31 degrees Celsius .
To test the stability of PHOCUS inside the locust, and instability outside of it, we have worked out which data needs to be collected to be admissible for field trials (see Field trials).
Phages rely on the metabolic processes of the host cell to enable viral replication. They are known to follow two possible life cycles; the lytic cycle and the lysogenic cycle . A lytic (virulent) phage does not integrate its genetic material into that of its host . While the life cycle of a lysogenic (temperate) phage consists of the viral genome, referred to as the prophage, being inserted into the genome of the host . PHOCUS will use a lytic phage, for several reasons:
- It will minimize the risks of horizontal gene transfer and/or phage mediated transduction . This could otherwise give certain bacteria an advantage, e.g. antibiotic resistance, and thereby disrupt the ecosystem . Given that our project focuses on lytic phages, generalized transduction is relevant. Generalized transduction involves the integration of random genetic information from the host chromosome into the phage. This rare event happens during phage replication with a prevalence rate of 1 in 11,000 phages, just before the viroid is enclosed inside the capsid of the phage .
- The lytic cycle ends with cell lysis and, therefore, cell death, reducing the chance of creating a new GMO.
- Due to the properties of the lytic cycle, the phages that reach their target bacteria will propagate quickly. This will lead to large increases in the amount of toxin and shRNA produced. When the host bacteria bursts, the Cry7Ca1 toxin, shRNA and RNAi associated proteins are effectively secreted, negating the issue of exporting the produced compounds out of the host. As the compounds are released in close proximity to the gut wall, they can easily reach their target. Higher Cry7Ca1 toxin and shRNA concentrations at the gut wall is correlated with a faster death rate, thereby making PHOCUS more effective.
Genetic engineering of the phage
We engineer phages to replace inessential genes with a gene coding for Cry7Ca1 toxin or shRNA. The risks involved with the spreading of engineered phages depend on the type of phage and the type of genetic modification. There are different concerns, apart from the previously mentioned horizontal gene transfer:
- Phage dissemination into the environment: Engineered phages with an expanded host range have a significantly increased change to spread into the environment . We decided upon using a phage cocktail, as there was not one single species of bacteria always present within the locust gut. With the use of this cocktail we spread multiple different phages, while these all separately have a chance to propagate. Increasing the total chance of propagation. Therefore we chose to engineer phages with a naturally narrow host range, so the propagation chance of our modified phage cocktail outside the locust is reduced.
- Production of harmful proteins: since phages can be engineered to express all types of proteins, the risks involved with the use of engineered phages strongly depend on the type of modification. We explicitly chose not to insert any sequences harmful for either the environment, humans or other animals.
- Persistence of the applied mutation in nature: To examine the stability of the mutation in engineered phages, the presence of the mutation can be determined over several generations of phages, as described by Nobrega et al. . In short, the engineered phage can be propagated in its host for several generations and the presence of the mutation can be confirmed by PCR after each generation. If our insert is extremely stable, our mutation could spread through genetic populations.
Toxin: specific, non-pathogenic, non-toxic for humans and other animals
Insecticidal crystal (Cry) toxins from Bacillus thuringiensis (Bt) are being used for biological control of pests worldwide, either through spray formulations based on spore-crystal preparations, or by introducing their genes (Cry protein or an active fragment of it) into transgenic crops. [20, 21]. In our project, we focus on the Cry toxin Cry7Ca1 identified in the B. thuringiensis strain BTH-13 that has been described to be effective against locust of the species Locusta migratoria manilensis, by puncturing the gut lining [22, 23].
Regarding safety we chose to use Cry7Ca1 due to different reasons:
- The Cry toxins are highly specific to their target insects, and therefore kill a limited number of species . Cry7Ca1 has been reported to have low or no toxicity towards Lepidoptera, Coleoptera and Diptera .
- Specificity of Cry toxins is provided by the midgut environment of the insect, that promotes toxin activation from the protoxin; and by the binding of the Cry toxin only to gut membranes that display the appropriate matched receptors .
- Humans do not have the same gut conditions nor the same receptors as insects. The Cry toxin should pass through us with no effect, being ingested like any other protein when eating food .
- These proteins have shown to break down in simulated digestive systems of humans . For allergen testing, we additionally tested the sequence of the Cry toxin, using the protocol from the Baltimore iGEM team of 2017, which came back as non allergenic .
Double stranded RNA: specific, non-pathogenic, non-toxic for humans and other animals
Our RNA of interference (RNAi) approach relies on the small interfering RNA (siRNA) processing machinery. We chose to use the RNAi approach because:
- siRNAs needs 100% sequence complementarity to the targeted messenger RNA (mRNA) in order to cleave them , ensuring extra specificity. Specificity is very important as any eukaryotic cell possessing active RNAi machinery is susceptible to silencing with our shRNAs.
- We can ensure specificity by choosing the right short hairpin RNA (shRNA) target. As phages will be sprayed in the environment, other species might be exposed to our shRNAs. The shRNA produced has to be screened for potential off targets to prevent fortuitous gene silencing in coexisting species in or nearby locust swarms, as well as humans and animals, through the use of transcriptomic databases. When choosing our target, we must ensure choosing specific targets, in order to prevent the surfacing of unwanted phenotypes, i.e. degregarisation  or increased food intake [31, 32].
Target bacteria: non-pathogenic, non-toxic for humans and other animals
For our biopesticide to work, bacteriophages that specifically target the bacteria present in the locust gut must be chosen as a delivery vector. Although the locust gut is not dominated by a single bacterial species, the desert locust maintains a constant population of the Enterobacter genus . To ensure sufficient toxin production, the biopesticide consists of a cocktail of phages targeting a variety of bacteria from the Enterobacter genus. Unfortunately, bacteria from the Enterobacter genus are not necessarily specific to the locust gut only. Although the Enterobacter genus is not a geographically conserved core member of the human microbiome, there are certain human populations that do show the presence of Enterobacter in their microbiome . Therefore, the possibility exists that our biopesticide could infect and kill the Enterobacter bacteria of certain humans or other organisms, while simultaneously producing the locust specific toxin.
However, these risks are small because:
- In humans, it is unlikely that the unintended killing of bacterial species by phages has negative consequences for human health, due to the strong selection pressure for non-pathogenic bacteria [S. Hagens, personal interview].
- For humans, the first line of defense against phages is the low pH in the stomach as most phages are sensitive to acidic conditions. .
- The risks for other organisms are also limited. Not only humans but also other organisms that contain Enterobacter in their gut [O. Lavy, personal interview] could potentially be at risk. However, the toxins that are produced are highly specific to locusts and should not harm those organisms. Nonetheless, to ensure our biopesticide is safe for other animals, more metagenomic data studies should be performed on different gut microbiomes.
Encapsulation: physical barrier
Encapsulation is a means by which the environment of a single molecule can be rigorously controlled . Several types of encapsulation exist, for example liposomes and polymeric microcapsules such as alginates .
- Encapsulation can act as a physical barrier to prevent off-target infection. The Enterobacter genus is known to prompt plant growth as they are involved in nitrogen fixation, soil phosphorus solubilisation and the production of antibiotics . A physical barrier is needed for our phages, regardless of the nature of the engineering we are performing, to avoid ecological imbalances on the vegetation the phage is sprayed upon.
A clear encapsulation method that meets our design requirements is difficult to find at first glance. Further research on encapsulation for our bacteriophages should be done.
Prevent resistance for toxin/shRNA
When exposed to pesticides, locusts with different phenotypic traits have a different chances of survival. Genes resulting in a higher chance of survival are more likely to be passed onto offspring. If this occurs for longer periods, pesticide resistant locusts can become dominant in a population. We have to prevent resistance for our pesticide to remain effective. The aforementioned traits include:
The Cry7Ca1 toxin attaches to receptors in the locust gut and pokes holes in the gut lining. Some locusts may have slight genetic alterations causing changes in the Cry7Ca1 receptor, resulting in inefficient binding, or none at all. Locusts with this trait will most likely pass it onto their offspring. The probability that this happens is difficult to estimate. Nevertheless, the risk that the desert locust becomes resistant to Cry7Ca1 is enhanced by:
- High exposure of locusts to Cry7Ca1 .
- Fast reproduction, resulting in many generations [39, 40].
The risk that this occurs is decreased by:
- The risk that this occurs is decreased by:
- Low initial frequency of phenotypic traits favoring survival .
Three different scenarios in which the desert locust evolves to become resistance to RNAi exist :
- The RNAi machinery of the desert locust is impaired due to mutations . However, an imposed RNAi system decreases chances of survival in a swarm due to virus susceptibility and, therefore, is not likely that this mutation propagates through the population.
- The target sequence in the desert locust is mutated to the extent that its mRNA is not sufficiently homologous to the siRNAs from the shRNA anymore . In case this would happen, it would be easy to switch to another target sequence of an essential gene.
- The desert locust may obtain mutations that decrease the stability of the shRNA in the gut or the uptake of the shRNA in the hemolymph . No estimate can be provided on the magnitude of this risk.
Two methods are being applied to mitigate the risk of the desert locust becoming resistant to PHOCUS:
- Two different modes-of-action: A chance exists that a desert locust is resistant against one of the two methods. The chance that a desert locust is less susceptible for both Cry7Ca1 and RNAi at the same time, however, is negligible.
- Monitoring: When PHOCUS is applied, the genomes of the desert locust should be monitored to assess whether a particular genetic variation becomes more prevalent in the population . If so, a strategy, such as tactically spraying PHOCUS in particular areas or by developing a novel toxin with a different mode-of-action, should be devised.
Prevent phage resistance
Bacteria evolve resistance to phages using many mechanisms . If the target bacteria are resistant to PHOCUS, the toxins are not produced to terminate locusts. We have specifically used lytic phages to lyse all the target bacteria to limit resistance development. Still, we studied the chances for resistance developing and came up with two scenarios in which phage resistance could occur:
- The first scenario consists of bacteria developing resistance to PHOCUS, starting with a non-resistant bacterial population. Based on our mathematical model, which studies the development of resistant bacteria, it is clear that our bacteriophage would infect and kill the entire Enterobacter population within a couple of hours - which prevents the bacteria to evolve resistance to PHOCUS.
- The second scenario consists of the existence of a resistant strain in the locust gut. Based on the advice of Dr. Steven Hagens, we have chosen to use a cocktail of phages that target different receptors of the same bacterium to address problems regarding resistant bacteria. This ensures susceptibility for our target bacteria.
Why is a kill switch unnecessary?
To reduce the risks related to GMO propagation in nature due to the release of the engineered bacteriophage, a strategy is to limit its spread in the environment . The amount of engineered phages in the environment will decrease naturally over time, making additional biocontainment measures uncalled for as:
- PHOCUS loses the competition with the wildtype phage for the host due to reduced fitness. Survival of PHOCUS depends on a selective advantage over the wildtype. However, the inserts do not confer a selective advantage to PHOCUS.
- The genetic inserts will be lost over time and the phage will return back to its wildtype sequence. This is due to the fact that transgenes often have a deleterious effect, a general consequence of disrupting the wildtype . We expect the relative fitness to decrease and hypothesize that this will result in the deletion of our insert, as is often the case for inserts in virus genomes .
- Bacteriophages are generally unstable when exposed to high temperatures and high doses of UV radiation. Different environmental factors can influence the stability of bacteriophages . Bacteriophages have difficulty with persisting on leaf surfaces because of inactivation by UV radiation [47, J.B. Jones, personal interview].
- The lytic nature of the engineered bacteriophage. A bacteriophage is dependent on the growth of their respective hosts to proliferate; they are self-limiting [48, 49]. This means that if a large amount of phages is released, their respective hosts will be locally eradicated. Limiting the spread and proliferation capability.
During interviews with experts in the field of, among others, phage biology and toxicology, potential safety concerns were mapped out and discussed. To address these concerns, risk mitigation strategies were developed and lab experiments were designed to test the safety of PHOCUS. We realized that, for PHOCUS to be used in the real world, it first has to go through field trials. To be admissible for these large-scale tests, more safety tests have to be done. Therefore, we investigated which data had to be gathered and designed the experiments accordingly, in addition to studying the corresponding legislation.
From our interview with Dr. Cecile van der Vlugt, senior risk assessor at the Dutch National Institute for Public Health and the Environment, we learned that even though legislation might be absent or non-permissive, the best approach to overcoming noncompliant legislation is by performing a risk assessment of PHOCUS. We most likely have to comply with three types of regulations:
- GMO regulations: Before taking a GMO into the environment, one needs to make a risk assessment for other organisms in the environment, e.g. we need to prove that the phage will not propagate indefinitely in nature.
- Insecticide regulations: Every insecticide needs to pass regulations before it can be applied. Answering questions such as; how specific is it? Does it persist in the environment?
- Food regulations: This will also apply to PHOCUS as it is a ‘living’ insecticide. Regulators will look at toxin/pathogenic effects of the modification made in the phage and how it could be passed to other organisms.
There are many different risks associated with the implementation of PHOCUS for which data should be gathered at a lab scale. This way we can prove the theoretical claims we have previously made, i.e. that PHOCUS is safe to humans. To move PHOCUS beyond the lab, many experiments must be performed to test its:
- Toxicity to non-target organisms (GM phage, Cry7Ca1, RNAi)
- Pathogenicity to non-target organisms (GM phage, Cry7Ca1, RNAi)
- Stability and potential to accumulate (GM phage, Cry7Ca1, RNAi)
- Uniqueness of sequence targeted (RNAi)
- Potential gene flow of insert (GM phage)
- Specificity to locusts (CryCa1, RNAi)
It should be noted that our perception of what is acceptable keeps changing. Therefore the list should be iterated over and altered after continuous discussions with risk assessors and managers. This is extra relevant in relation to our project, as we have a novel application and therefore there may be potentially unidentified risks that could surface over time.
After completion of the lab-scale risk assessment and acceptance by the responsible authorities, PHOCUS can move to field trials. Here, PHOCUS will be subject to different tests in an outside environment, i.e. an environmental risk assessment (ERA) . The experiments performed could be the outside variant of the ones performed in the lab. This data has to be generated again as the actual environment is much more complex and could lead to different interactions. The specifics of what data exactly needs to be gathered remains imprecise. Primarily because our approach is novel and the responsible authorities have not yet drawn up the related legislation. They will likely do this using a case by case approach, as advised by scholars [52, 53]. Still, it will likely remain comparable to existing ERAs for GMO, pesticide and food regulations, e.g. the ERA for GM plants in Australia,which looks at "toxicity, allergenicity, nutritional profile, agronomic characteristics, increased disease burden, spread and persistence of the GMO, gene flow etc." . Further directives for the use of biological control agents are available . While critique of the contemporary ERA on GMOs, which use Cry toxins in transgenic plants, is also presented , it becomes clear that experts on risk assessment do not agree on the best approach. Additionally, different countries also have different procedures. Therefore, it is essential to understand that there is not one set way in which PHOCUS will gain approval in all countries, and its regulations should be assessed in close collaboration with the responsible authorities of the affected countries.
After the risk assessments have been conducted, it is important to submit these results to the Pesticide Referee Group (PRG). This is an independent group of scientific experts on pesticides. They assess the data submitted and create a shortlist of pesticides they deem acceptable. This list is specified for its intended use. The FAO advises countries on which pesticides to use. They only buy and advise pesticides that have been shortlisted by the PRG. Lastly, all countries have their own legislative freedom, therefore PHOCUS would still need to be admitted in every country individually.
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