Team:TUDelft/Safety
Safety
To ensure safe development and implementation of our biosensor, AptaVita, risk assessments were performed in- and outside the laboratory, as well as analysis of the intended implementation site. Here, we present these risk assessments, amongst others the Safe-by-Design strategy, that were used to identify and analyse risks associated with our product AptaVita.
Safety
Safety is an important aspect to consider when our vitamin biosensor is implemented in society and within our work performed in the laboratory. As synthetic biology is still in development, we aim to ensure safety as much as possible. Therefore, we analyzed safety, risks, and ethical issues that arise in our project. Risk management can be split into biosafety and biosecurity. Biosafety aims at protecting public health and the environment by ensuring safe working practices and anticipating (uncertain) emerging risks associated with biological agents to prevent accidental exposure [1]. Biosecurity aims at the prevention, control, and management of misuse through loss, theft, or intentional release of any biological agents [2]. On this page, you can find more about Safe-by-Design - an iterative risk management approach -, biosecurity, and lab and biosafety regarding the AptaVita project. To find out more about the implementation of the biosensor in Uganda and the safety considerations for implementation, visit our Proposed Implementation page.
Safe by Design
We choose to apply the Safe-by-Design approach to identify and anticipate possible risks within AptaVita, taking biosafety and -security aspects into account. We performed this Safe-by-Design approach by considering the full process from project design to project implementation to make the AptaVita device as safe as possible. We received help from the National Institute for Public Health and Environment in the Netherlands (RIVM), who guided us in setting up the Safe-by-Design of our project. Fig 1. gives a preview of the aspects discussed in our Safe-by-Design for the different AptaVita stages.
Biosensor design
AptaVita is designed to be a biosensor that is able to detect vitamin deficiencies in blood samples via the expression of a genetic circuit regulated by a vitamin binding aptazyme. The read-out of the genetic circuit is based on the expression of a reporter gene that generates a colorimetric read-out by substrate conversion. Already at the design stage of the biosensor, several safety aspects and potential risks are considered and identified:
- The usage of genetically modified organisms (GMOs) includes biocontainment risks due to the reproduction opportunities for these organisms in the environment [3]. To prevent this risk, one of the biggest biosafety-design decisions we made, is to employ cell-free systems to avoid the usage of GMOs in the device and to ensure that the biocontamination risk is as low as possible.
- We considered LacZ and XylE as reporter genes that facilitate the colorimetric read-out by converting their colored substrates, respectively chlorophenol red-b-D-galactopyranoside (CPRG) and pyrocatechol. However, due to the toxicity and hazardous risks of pyrocatechol, we decided to continue with LacZ as a reporter gene as CPRG is a safer chemical to use [4].
Manufacturing
Several safety aspects should be considered at the manufacturing stage of the biosensor. The AptaVita detection system consists of several parts, which can be categorized into hardware and biochemical components. The biosafety- and biosecurity-related issues apply mainly to the biochemical components, namely to the plasmids containing the reporter gene and the cell-free system. Both have to be manufactured at a large scale for the biosensor to be commercialized. Although we avoid using GMOs in the device itself, we are restricted to E.coli bacteria for the amplification of the genetic material containing the reporter gene. Therefore, local GMO regulations should be taken into account to make a proper decision about the location of manufacturing. We consider the Netherlands and Uganda as our two options for localization of production, because the Netherlands and Uganda are respectively the countries in which the AptaVita idea is designed, and the device is applied.
GMOs for scaling up plasmid production
One part of manufacturing AptaVita includes the scaled-up production of the plasmid containing the aptamer within modified E.coli. This work with modified E. coli has to adhere to GMO regulations that apply in the country of production. Dr. Martin van Gijzen mentioned that trustworthiness issues could potentially arise from local production of our vitamin detection test in Uganda [TU Delft, interview Martin van Gijzen]. However, this was contradicted by Tosca Terra from the Healthy Entrepreneurs and the outcome of the village healthcare worker survey [TU Delft, Healthy Entrepreneurs, VHT survey]. Therefore, we concluded that trustworthiness issues would not be a major problem for local production. However, Atek et al. showed that biosecurity and biosafety measures in Uganda are currently not robust enough and resulted in a biosafety biosecurity performance of only 33% for both the private and public healthcare sector [5]. This is in agreement with the Global Health Security Agenda (GHSA) assessment for biosafety biosecurity performance which was determined at 41% for many facilities. These laboratories often remain inadequate in terms of biosafety and biosecurity systems, occupational health and safety strategies, safe waste disposal mechanisms, and infection prevention and control mechanisms [6]. Nationwide attention to biosafety and biosecurity would be required to stimulate biosafety and biosecurity measures.
Due to a lack of clear biosafety and biosecurity legislation in Uganda, we have decided on the production of AptaVita in the Netherlands for now, which mostly follows the GMO legislation of the European Union (EU). Within the EU, strict measurements have been taken to ensure biosafety, which EU countries must implement into their own legislation. Most of the GMO regulations in Europe can be found in the Dutch GMO regulations [7], which are applied to assess the risk, pathogenic class, and biosafety level of the bio components that are used within our device. Even though EU legislation has to be followed for production, the implementation site is envisioned in Uganda. Thus, we also have to adhere to the regulations in Uganda.
Production of the cell-free system in bulk
Besides the genetic construct, the cell-free system is another key aspect of AptaVita that should be manufactured on a large scale. The cell-free system could be produced based on the homemade cell-free system, OnePot PURE, a protocol that the Overgrad Grand Prize winners, iGEM EPFL of 2019, successfully performed [8, 9]. The previously mentioned GMO measurements also apply for the bulk production of the cell-free system needed for AptaVita, as E.coli strains are required for the production of the cell-free system.
Transport
Risks should also be minimized during transportation of the biosensor. Based on the study of Pardee et al., we chose to freeze-dry the paper-based test containing the genetic construct, cell-free system, and CPRG substrate. The freeze-drying increases the biosafety of the biosensor during transportation and ensures a sterilized and abiotic test during transportation. A healthcare worker can subsequently rehydrate this freeze-dried test by adding a patient sample onto the biosensor. In addition, no cold chain transport is required as the freeze-dried devices can be stored at room temperature [10].
Usage
For the usage of AptaVita, there are various safety aspects and risks to consider.
Gain-of-function by ampicillin resistance genes on plasmid
One of those safety aspects includes the test containing a plasmid with an ampicillin resistance gene, necessary for amplification during biosensor manufacturing. The ampicillin resistance gene could potentially give an evolutionary advantage, therefore resulting in plasmid uptake by microorganisms when the biosensor is utilized outside the ML-1 designated laboratory spaces. Hence, the microorganisms could get a gain-of-function such as resistance against antibiotic medication. This mainly means that we need to be careful with disposal, which can be read in one of the next sections.
Risk of spreading blood infectious diseases
Furthermore, blood withdrawal with a finger puncture is required as a sample for our biosensor, and this brings the potential risk of spreading infectious diseases. Therefore, obtaining the blood samples should adhere to the WHO guidelines [11]. This means that manuals should be distributed, and trained personnel should perform the blood withdrawal with sterile needles. To avoid this risk and ensure the safety of both patients and personnel, we have decided on implementing AptaVita within healthcare facilities. The full argumentation why we have chosen for the implementation of our biosensor in hospitals can be read in the Proposed Implementation page.
Unauthorized reselling of biosensor
We intended to design a biosensor that will be used by official organizations such as health care facilities. Distribution of the biosensor to other individuals/parties that are initially not well informed or advised about the biosensor can lead to safety issues. These safety issues can be: an increased spread of infectious blood diseases, or incorrect disposal of the diagnostic test when carried out in a high blood infection disease environment.
Misuse of personal data
From the dual-use quick scan, it became evident that the dual-use character comes from the vitamin deficiency database. Especially in the current era of rapid implementation of mobile and digital tools in the health sector, the risks of privacy issues in health databases have increased [12]. Therefore, it is essential to determine the risks associated with the vitamin deficiency database. The leakage of data could result in an increased potential for discrimination. The misuse of this health data could give rise to discrimination determining someone’s eligibility for employment, housing, or other services. A micronutrient deficiency can indicate the chances of someone getting ill and, therefore, be unfavorable for a future employer. This risk could be heightened in low-resource settings due to intersectional inequalities, governments that are lagging in the implementation of data protection policies, and where regulations for protecting personal information are missing. However, the Uganda Ministry of Health is aware of issues regarding the misuse of digital health data. Therefore, the government has set up the Uganda National eHealth Strategy 2017-2021 [13].
Besides the government taking measures to reduce the risks regarding health data privacy, we also took measures to minimize the risk of misuse of personal health data. As the risk of data spreading is especially increased when using personal computers [12], we decided to adjust our read-out method to a dedicated hardware device. The dedicated hardware device will collect the patients’ data and subsequently upload the data to the vitamin deficiency database, thereby circumventing the usage of personal digital devices [TU Delft, interview of Dr. Diehl]. Furthermore, to prevent privacy issues, the data will be stored and shared anonymously with the health organizations that collect the micronutrient deficiency data. The only information required will be age, gender, and if a vitamin deficiency was detected. The patient will receive treatment directly from the doctor. Therefore, the stored data will only be relevant to determine high-risk areas that require more attention to prevent vitamin deficiencies.
Disposal
Given the purpose of our biosensor to generate health data and the usage of a blood sample for this test, the detection device to be developed must be a single-use one and cannot be recycled. This means that a considerable amount of waste can be expected to be generated from this application. Therefore correct disposal of the device is crucial.
Proposed disposal of the AptaVita device
A disposal manual should be included to ensure that the biosensor waste is disposed-off correctly to minimize the biocontamination of the genetic material and to avoid the spreading of infectious diseases. Dedicated bins should be provided with the biosensor to ensure that all biological material is collected and treated in the same way. Although the disposal regulations in Uganda are not as extensive as in Europe, there are guidelines for the development and implementation of health care waste management, which are supposedly integrated into the health care facilities [15]. We can take advantage of the current existing disposal infrastructure within health care facilities to ensure the safety of the patients and personnel.
Biosecurity
Dual-use
iGEM values safety and biosecurity, and explicitly states that teams are expected to manage any risks. Amongst these risks, iGEM proposes that the risk of misuse can be managed through dual-use practices [16]. Potential misuses of biosecurity are difficult to imagine, as one may be limited to their own worldview when considering potential uses. To guide us through the identification of possible good and bad uses of our product, we consulted the Bureau of Biosecurity from the National Institute for Public Health and the Environment in The Netherlands. The Bureau of Biosecurity developed a tool for dual-use research of concern evaluation. This tool, called the Dual-Use Quickscan, consists of a questionnaire of 15 questions and is developed to identify dual-use of the product and to contribute to the general awareness of dual-use. These questions are extracted from scientific literature and focus on safety and biosecurity. One can respond with either "Yes", "No", or "unknown". Depending on our responses to these questions, the possibility of a dual-use character is calculated based on relevant literature [17].
According to the Bureau of Biosecurity, if one or more questions are answered with "Yes" or "Unknown", our device could have a dual-use character [18]. Concluded from this quick scan, two questions were answered with a "yes" response and therefore identified as risks within our project. One of them entails the biocontamination of our plasmid into the environment. Although we are using a GMO-free biosensor, releasing the plasmid in the environment could still result in biocontamination. The second identified risk entails the knowledge and power misuse that comes forth from the vitamin deficiency database. Both risks are further elucidated in the Safe-by-Design section.
Lab safety
General Safety
Our iGEM team's laboratory is located at the Bionanoscience department of the Applied Science faculty at TUDelft (Fig. 2). The laboratory is in full compliance with the ML-1 lab requirements and the iGEM safety and security policies. We designed all our experiments to be done at the ML-1 biosafety level and solely utilized microorganisms classified in this risk group. To reassure that our team members have the required knowledge for emergency operation procedures and to perform the laboratory work safely, all of our team members passed the following safety tests:
- General building safety (meeting points, emergency numbers, flight plans, etc.).
- General laboratory safety (chemicals, waste disposal, clothing, safety precautions, etc.)
- Biological safety (ML-1 grade safety, biological waste, safety precautions, etc.).
- Laser safety (general safety precautions).
In addition, team members that contributed to the wet-lab work completed lab training given by one of our supervisors. Within this lab training, we also learned how to discard different types of waste in the correct disposal bins. Before being allowed to perform any experiments in the lab, a general safety report covering all the involved activities, chemicals, organisms, and other relevant safety information was handed in and received approval by the Institution's Biosafety Officer. All the acquired biological parts (genes, plasmids, strains) were first registered in the Institution's local repository and received approval before being ordered.
Safety regarding COVID-19
To reassure the safety of our team members regarding COVID-19, the following measures were implemented:
- Team members stayed home when they were suffering from symptoms related to COVID-19 following the rules of the Dutch National Institute for Public Health and the Environment (RIVM).
- Within TUDelft several hygiene rules and cleaning measures were followed, such as frequent cleaning of hands, workplaces, and toilets.
- Team members and other people entering the building were obliged to wear face masks when transferring from one place to the other.
- Due to the corona restrictions, only a total of four students were allowed to be present in the lab at the same time. Prior to the week, we made a schedule of the experiments to clearly show who is present in the lab at what time and to reassure that the person limit in the lab is never exceeded.
Biosafety
Strains
AptaVita is designed to be GMO free. E.coli top10 is solely used for storage, construction and amplification of the genetic constructs used in the biosensor. This E.coli strain can be utilized at ML-1 biosafety level.
Cell-free system
Cell-free systems with a prokaryotic (E.coli) and eukaryotic (Wheat Germ) background are used in our experiments. These cell-free systems contain cell-free components of enzymes involved in transcription and translation and originate from ML-1 E.coli and Wheat Germ strains. These components, on their own, pose ML-1 risks to humans such as when ingested or by skin exposure for a considerable amount of time. By following the ML-1 lab protocols and regulations, we avoid possible harm to laboratory staff.
Vectors and inserts
The plasmids that were used, were ordered at Twist Bioscience. The plasmids contain a lacZ reporter gene under the control of a T7 promoter and terminator that can be expressed in a prokaryotic E.coli cell-free system. Furthermore, the plasmids contain an aptamer in the 5’UTR or 3’UTR that will control the expression of the reporter genes as well. The backbone of the three plasmids consists of the pTwist Amp High Copy vector, which contains a pMB1 origin and a gene for ampicillin resistance in E.coli. All features of these plasmids are in compliance with the ML-1 regulations.
Chemicals
We use some chemicals that are hazardous, such as SYBR Safe. The incorrect handling of carcinogenic and toxic materials could result in the exposure and negative health impact of the users and third parties. Additionally, incorrect disposal could result in a risk for society and the environment. Therefore, all of the material handlings are to be performed with adequate gloves and visual aid on the correct disposal flow is available in the area for correct waste management.
References
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- International Food Safety Authorities Network. (2010, March 3). Biosecurity: An integrated approach to manage risk to human, animal and plant life and health. https://www.who.int/foodsafety/fs_management/No_01_Biosecurity_Mar10_en.pdf
- Rijksoverheid. Beleid genetisch gemodificeerde organismen. Retrieved September 10, 2021, from https://www.rijksoverheid.nl/onderwerpen/biotechnologie/beleid-genetisch-gemodificeerde-organismen
- U.S. Environmental Protection Agency (2000). Catechol (pyrocatechol). Retrieved September 16, 2021, from https://www.epa.gov/sites/default/files/2016-09/documents/catechol-pyrocatechol.pdf
- Atek A.K., Owalla T.J., Baguma A., Okwalinga P., Opio J., et al. (2018). Biorisk Management Practices in Public and Private Laboratories in Uganda: A Nationwide Baseline Survey. Journal of Bioterrorism and Biodefense. 9: 164. doi:10.4172/2157-2526.1000164
- Central Public Health Laboratories of the Republic of Uganda. (2021, October 1). Infrastructure, Biosafety and Biosecurity. http://cphl.go.ug/about-us/nhlds-units/facilities-and-safety
- Regeling genetisch gemodificeerde organismen miliebeheer. (2013). https://wetten.overheid.nl/BWBR0035072/2020-04-01
- EPFL 2019 iGEM team. (2019). OnePot PURE. https://2019.igem.org/Team:EPFL/OnePot_Pure
- Lavickova, B. & Maerkl, S.J. (2019). A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System. ACS Synth. Biol., 8(2), 455–462. https://doi.org/10.1021/acssynbio.8b00427
- Pardee, K., Green, A. A., Ferrante, T., Cameron, D. E., DaleyKeyser, A., Yin, P., & Collins, J. J. (2014). based synthetic gene networks. Cell, 159(4), 940-954. doi:10.1016/j.cell.2014.10.004
- World Health Organization. (2010). WHO guidelines on drawing blood. Retrieved 10 september, 2021, from https://www.euro.who.int/__data/assets/pdf_file/0005/268790/WHO-guidelines-on-drawing-blood-best-practices-in-phlebotomy-Eng.pdf
- Tiffin, N.,George, A.& LeFevre, A.E. (2019). How to use relevant data for maximal benefit with minimal risk: digital health data governance to protect vulnerable populations in low-income and middle-income countries. BMJ Global Health
- Republic of Uganda Ministry of Health. (n.d.). Uganda National eHealth Strategy 2017-2021. Retrieved 16 September, 2021, from https://health.go.ug/sites/default/files/National%20e_Health%20Strategy_0.pdf
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