“Better safe, than sorry”
It is crucial to pay attention to the safety and security of IBDetection. The ultimate goal is to produce and use IBDetection in a safe manner. Here, we describe the lab and project-specific safety measures as well as the applied Safe-by-Design approach that encouraged us to reflect critically upon the hazards, risks, and risk reduction measures throughout the project and the future of IBDetection.
Regular safety measures in the lab
Biosafety is an important concept that has to be considered before performing any kind of biological research with pathogens or Genetically Modified Organisms (GMOs). Considering this, one should think about the possible hazards of these organisms and take appropriate measures. Therefore, biosafety consists of all containment principles, technologies, and practices that are implemented to prevent unintentional exposure to pathogens and toxins or their accidental release.
Our team consists of eight master students, who have already some experience inside different types of laboratories. Therefore, we all had several safety instructions and performed supervised experiments of different kinds including working with chemicals and with GMOs. Nevertheless, we had the lab introduction again to get ourselves informed about all current measures. Especially, the biosafety measures at the Eindhoven University of Technology (TU/e) and the measures regarding the Microbiological lab I (ML-I lab) were repeated. This started with a guided tour in the ML-I lab and a Safe Microbiological Techniques course from the Biosafety Officer of the TU/e, in which we learned the importance of safety, what the Dutch and European legislations are regarding GMOs, and precautions taken by the TU/e for working safely with GMOs. Furthermore, we discussed standard measures, such as wearing a lab coat, keeping windows and doors closed, and neither eating nor drinking in the lab. The three most important subjects from the SMT course were: 1) the disposal of (non-)biological waste, 2) how to work safely with GMOs in the lab, and 3) how to assess the risks of research. In the end, we applied this new knowledge in theoretical cases from the BSO and received SMT certificates.
Project-specific safety measures
Beside the regular precautions, we also need to assess the specific risks of our research. The safety form was a useful tool to help us with the risk assessment. The most important details will be further elaborated below.
As explained in the project description, we will test our design in E. coli BL21 (DE3) cells. According to the Dutch legislation , these cells are a type of E. coli that has been altered to be safely used (for research) inside an ML-I lab. Therefore, it is “very unlikely to cause disease in humans, animals or plants” . Most of the experiments have been done with the BL21 (DE3) cells, however, some experiments have been done with other types of altered E. coli, which are used for plasmid stability and molecular cloning applications. Other types of cells used are the DH5 alpha cells, in which the ordered plasmids (Addgene) have been delivered.
Furthermore, XL10 Gold, Novablue, and TOP10 cells were used, in which, after restriction, ligation, and mutagenesis reactions, several plasmid amplifications have been performed. By transforming the plasmid DNA into these cells, growing a culture from a single colony, and extracting the DNA using a Miniprep, the extracted DNA was then used for further experiments in BL21 (DE3). Moreover, Novablue , TOP10, and DH5 alpha cells were used for part improvement.
Additionally, the risks of our plasmid and the corresponding proteins need to be assessed as well. For the proof-of-concept, we used three different plasmids, which all have been ordered from Addgene. These are the pKD227, pKD233-7.3, and the pET28a_T7-ARG1. The first plasmid is a p15A backbone containing a spectinomycin resistance, a LacI sequence, and the TtrS sequence, which produces the membrane protein required for our sensor. The pKD233-7.3 is a ColE1 backbone, containing the TetR sequence, required for the TtrR protein and regulated by the pLtetO-1 promoter. The TtrR protein is the regulator protein in our sensor,
which activates the pTtrB165-269 promoter in front of the GFP and this protein is also present on the pKD233-7.3. Besides, the plasmid also contains a chloramphenicol resistance and a constitutively expressed mCherry. At last, the pET28a_T7-ARG1 plasmid is, as the name suggests, a pET28a vector, containing all 12 ARG1 proteins, regulated by a T7 promoter. Furthermore, it contains a LacI sequence, a F1 origin, and a kanamycin resistance. To be able to combine the sensing part with the reporter part of our sensor, we need to change the T7 promoter into the pTttrB165-269 promoter, which makes it responsive to the TtrR protein. Fortunately, all of these backbones, inserts, and proteins are non-toxic and are part of the GMO license of the TU/e, and can be used in an ML-I lab.
At last, the risks regarding the handling of all chemicals and organisms inside the lab are at least as important as reducing contamination of your experiment and as minimizing hazards. Therefore, we prepared all experiments based on protocols from various literature resources [3-5] and our supervisors. In addition, dr. Yan Ni assisted us during experiments that we had never performed before. When we performed the protocols and knew the risks, it was no obstacle to perform the experiments ourselves. Once something of the protocol was unclear, a more experienced person (PhD or higher degree) was available in the lab to give us advice.
When working with different types of chemicals, we considered all of them and their toxicity in advance, and used the corresponding precautions (e.g. gloves or fume hood). As explained above, we always followed the lab rules, wearing goggles, and a lab coat in the lab at all times.
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As described on Lab Safety, working in the lab with GMOs comes with many safety concerns. However, the safety concerns related to the application of the GMO itself should also be properly evaluated. GMOs can be used for various applications, all coming with their risks and hazards. With IBDetection, the GMOs are used for the monitoring of IBD inside the human body, making elaboration of the safety issues even more important. This was emphasized during the patient interviews. When we asked them if they would ever consider ingestion of a pill containing GMOs, we got the following answer:
“Only when it is completely safe for human beings and the environment.”
“Only when the pill is extensively tested in clinical trials to assess all possible risks.”
The patient interviews made us realize that it is very important to elaborate on the potential hazards and options for risk reduction related to our project. We got the opportunity to participate in the Safe-By-Design assignment, a safety approach that encourages critical reflection on hazards, risks, and risk reduction measures early in the innovation process, of the Dutch National Institute for Public Health and the Environment (RIVM) . Safety is paramount, and should be continuously addressed during the design process, especially during conceptualization, and when theorizing application of the final product. We spoke to three RIVM experts and discussed our project concerning safety and ethics. They encouraged us to identify all possible hazards that arise when our pill might reach the market in the future. Some of the hazards we came up with are:
Misuse: the pill can fall into the hands of malicious individuals and entities.
Subject safety: the bacteria influence the already disturbed microbiome of IBD patients.
Environmental safety: the bacteria are released during production, distribution, and usage into the environment.
Horizontal gene transfer: the conjugative plasmids can introduce new genes into the genome by replacing existing genes.
The above-identified hazards will be described in further detail in the following paragraphs.
If the pill is misused, either intended or unintended, it might pose a biological threat to public health and national security. This is called dual-use of the product. Intended misuse can arise when the plasmid is altered in a way that it produces harmful proteins inside the human gut once it detects tetrathionate. In this way, the pill will be dangerous for the users, i.e. patients suffering from IBD. However, this intended misuse of our product is very unlikely as altering a plasmid requires biological knowledge,
a lab, and harmful DNA sequences. Unintended misuse can arise when the pill is accidentally swallowed by someone, for instance, a child. This will be highly unlikely as the pill will be prepacked and the package will clearly describe what is inside. With this reasoning, we can conclude that the hazards related to dual-use of our product are limited.
To put into perspective the full extent of the effect of our GMO on the human gastrointestinal tract (GI), e.g. causing disturbance of the microbiome, we got the gracious opportunity to test the bacteria on our partner iGEM team BOKU Vienna’s gut on a chip. Unfortunately, we did not get the chance to send our GMOs to them in time. Nevertheless, from the literature, we found that the E. coli Nissle 1917 strain we intended to use for our proof-of-concept has potentially been associated with cancer development and inflammation flare-ups [2,3]. Our hypothesis is that this will be confirmed by testing it on the gut-on-a-chip. Therefore, we should look into the possibility, applicability, and consequences of using other cell lines as candidates for our product, e.g. L. plantarum WCFS1 (LPW) . The LPWs are already used for genetic research, have probiotic properties, and are present in the human gut microbiome . Therefore, LPWs are a promising candidate to implement our system in, for future research. As the LPWs will still be modified, they might pose hazards to the human gut such as disturbance of the microbiome. It is, therefore, highly recommended to test GMOs on the cells of interest, to identify these potential risks and indicate whether or not other microbial cell lines need to be used. Next to that, the effects of the LPWs may vary from person to person. We discussed this point with team iGEM BOKU Vienna and we concluded that it was necessary to research this thoroughly. This should not be a problem since this is also part of the standard clinical trials of the medical examination .
When GMOs survive at unwanted places, they may cause significant trouble. Therefore it is of high importance to regulate when and where the GMOs are alive or not. To do so, a kill switch can be incorporated into the plasmid design. Kill switches are genetic circuits that, depending on the environmental conditions, selectively eliminate GMOs. In this way, it can be ensured that bacteria are only alive in a defined area, in our case the human gut and inside the pill, and can (theoretically) not escape into the environment. Kill switches can be based on several environmental conditions such as pH and temperature [7,8]. To assess which kill switch suits our system best, we must first identify where we want our GMOs to function, and where they cannot survive. We thought of the specific environmental conditions that are present inside the gut of IBD patients. The first thing we came up with was the high variety of pH levels inside the GI tract. The pH in the terminal ileum and colon is in general higher than in other regions of the GI tract. Therefore, a pH-dependent kill switch was our first option. However, this kill switch was soon rejected as the pH levels vary a lot inside the colon of IBD patients . Then, we thought of the bacteria already living inside the human gut. Knowing that the gut microbiota largely consists of anaerobic bacteria (over 90%), it was deduced that our GMO should fit these oxygen-deprived conditions . As the environment is rich and the human gut sparse in oxygen, a kill switch dependent on the oxygen level might suit our project. We found an article in which the authors studied the “cytochrome o and d oxidase gene expression” in E. coli under anaerobic and aerobic conditions. It turned out that the organism was able to survive better under oxygen-deprived conditions, as is found in the human gut . Therefore, the oxygen-dependent kill switch is a potential candidate that can be incorporated into the plasmid design in future research. This kill switch reduces hazards by ensuring cell death once the bacteria are released into the outside world. Due to time restrictions, we were not able to do it ourselves.
In addition, the production process of the GMOs, as well as the pill should be considered. GMOs are used in various laboratories and must be produced in a safe environment such as a bioreactor. Furthermore, it is essential to implement the industry-accepted Good Manufacturing Practices to maintain efficacy and safety of the pharmaceutical products, while keeping the GMOs alive, i.e. not activating the kill switch. iGEM team BOKU Vienna’s came up with the idea to dry the GMOs and allowing them to go in a resting phase. By doing so, the GMOs can be stored at room temperature under a vacuum to prevent them from being moist.
Horizontal Gene Transfer
Horizontal gene transfer (HGT) is the non-sexual movement of genetic information between genomes of different hosts. Sharing DNA or RNA between bacteria is a common occurrence that results in the introduction of new genes into the genome by replacing existing genes . Plasmids are important drivers of HGT and can transfer genes at high rates . In our project, we introduce two different plasmids to create our GMOs. These GMOs are released in the human gut where a lot of different microorganisms are present. Therefore, the risk of HGT between bacteria cannot be excluded. One of the main concerns here may be the transfer of antibiotic resistance genes of our plasmids towards the microorganisms in the microbiome. This would result in antibiotic-resistant bacteria which should not be resistant. If infectious bacteria become resistant it may result in infections that cannot be cured with regular drugs/antibiotics. A solution for this might be the stable integration of our designed DNA into the bacterial genome. There are certain techniques that can do this, but for the scope of our practical research, this was not doable due to the limited amount of time .
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