Team:Thessaly/Safety
SAFETY.
TU Delft 2018 team was right when they preferred using “safety always” than “safety first”. Safety should accompany every iGEM project throughout its development considering not only laboratory safety but ethics too. For this reason, during our whole project we tried to incorporate safety measures respecting society, environment and ourselves.
Choosing a project that aims to diagnose Tuberculosis (TB), we should take strict safety measures since M. tuberculosis (MTB) is a Risk Group 3 Organism. Furthermore, ODYSSEE is designed for a field application, so personnel using our test as well as patients being diagnosed are inevitably exposed to its components. Thus, it was of high importance for us to ensure that safety policies are kept strictly throughout the whole procedure, while minimizing the risks of human error. Aiming finally at applying our test in refugee camps in Greece, as well as worldwide, we conducted our Human Practicies activities with great consideration of ethics and safety.
Lab Safety
Safety in our university
Project safety and prevention of hazardous situations is an important aspect of our concerns, but personal safety and proper use of equipment is a critical matter as well. During our project, we received extensive guidance and supervision regarding equipment usage (like autoclaves and laminar flow hoods) in the lab by our instructors and staff scientists to ensure that accidents will be promptly avoided.
Images 1-2. Our laboratory facilities
Moreover, it’s always informative to study the Safety Pages online about biosafety knowledge and issues, but a talk with someone experienced on biosafety is always a good thing. Before getting into the lab, in order to have inclusive knowledge on our University’s regulations and rules, Dr. Tilemachos Dimitriou, Post-Doctoral Fellow at the Molecular Virology – Molecular Bacteriology lab, prepared a comprehensive presentation on the Biosafety policies of our University and gave us extensive information on Biosafety levels. We also discussed potential safety issues for our project based on the design we had at the time.
Images 3-4. Lecture on Biosafety and Safety regulations by Dr. Tilemachos Dimitriou
Chassis Organism
Because Mycobacterium tuberculosis is a Risk Group 3 Organism, we could not handle it according to iGEM’s Safety regulations and policies. To circumvent this problem, we are using a chassis organism to conduct the experiments in a faster and much safer manner. Our chassis organism is the common lab bacterium Escherichia coli which has been studied for years and the risk for a harmful response is minimal and easily calculated. Strain DH5-alpha, a derivative from K-12 strain, is our go-to strain for plasmid propagation and isolation, while strains BL21 (DE3) and M15-T7 deriving from E. coli strain B, have the role of “protein factories” enabling protein expression of the reporter genes needed to visualize the produced signal from biomarker detection for our in vivo experiments. As soon as we decided which biomarker we would use, we contacted iGEM’s Safety Committee (Feb 26, 2019) to verify that we were able to work under those conditions. The immediate response was a green light for us, meaning we had designed our project in a safe manner.
Design of Biosafety
ODYSSEE was designed to ensure safety for potential users and the environment. The main safety pillars of our project design are discussed below.
Cell-free system
Genetically Modified Organisms (GMOs) and their release in their environment are controversial topics within the scientific community. Nonetheless, in accordance with iGEM’s safety rules and standards, we do not plan to release GMOs in the environment where they could possibly cause imbalances in the habitat’s ecological stability. To avoid any such hazardous occurrence, we are using a cell-free system to diagnose Tuberculosis, instead of live bacteria. The cell-free system does not involve any potentially harmful biological parts and its sub parts are approved and classified as nonhazardous. Moreover, since the tools we have implemented in the cell-free system are orthogonal, there will be no gene expression of the reporter gene for signal producing when the unique synthetic activator we have designed is absent, even if those parts are accidentally released into the environment.
Biological sample
The most important factor of a diagnostic test considering its usefulness and safety, is the biological sample that is going to be used for the detection of the microorganism. Sputum and blood are the commonly used biological samples for TB diagnosis, using DNA/protein markers or via culturing the bacteria in plates with the appropriate medium. Using sputum as a biological sample implies that alive bacteria exist in the sample, and thus may potentially transmit the disease in case of wrong sample treatment. Furthermore, blood sampling is considered to be an invasive method endangering the patient’s approval in such a test.
In order to overcome these implications and design a biosafe test, ODYSSEE uses urine, an accessible, non-invasive fluid as the biological sample. MTB DNA is found in patients with active TB, in the form of cell-free DNA (cfDNA). cfDNA consists of the remnants of the MTB genome which after cell death end up in the patient’s urine, in the form of DNA fragments. These fragments are harmless and non-pathogenic, providing a safe way to detect TB.
Biomarker
The biomarker is the second most important factor for the accurate and reliable diagnosis of all diseases, including TB. Diagnostic biomarkers for deadly and serious diseases, like Tuberculosis, ought to be selected based on strict criteria considering their toxicity and harm both for the society and the environment.
Our biomarker is a DNA fragment of the MTB genome found in urine. The fragment we selected to use is the IS6110 gene, which is highly conserved within the MTB complex (which causes TB) and present in multiple copies in the bacterial genome. IS6110 is an insertion element found exclusively within the members of the Mycobacterium tuberculosis complex (MTBC), and encodes for proteins responsible for the transposition activity that occurs naturally in a wide variety of organisms. However, unlike other transposons, IS elements including IS6110 do not contain accessory genes for antibiotic resistance. The protein encoded by IS6110 is a transposase only. Hence, it is not involved as a causing agent in the disease itself. Furthermore, for safety reasons regarding the transposase that IS6110 produces naturally, we cloned (into E. coli) and used in our experiments only a non-functional fragment of this part. Expression of the transposase is prevented, because the promoter-like sequence that exists in the 5' end of the IS6110 is absent in our cloned fragment, and thus transcription/translation is unable to occur. This aims to avoid the transposition of sequences of GM strains to wild type ones outside the laboratory, ensuring the highest level of biosafety.
Product Design
In order to carefully implement our technology into a final productwe developed the “2tubes Philosophy”, a vision to transform the complexity of our lab’s experimental steps into a solution ready to be applied in limited resource settings. Designing a biosafe product both for the user and the environment was our top priority. For this reason, our product’s package is supplemented with detailed information about the product and the protocol that should be used in order to execute the test. While doing the test, we recommend using gloves and dispose the remaining tubes in a biohazard waste bin. Finally, our product is designed with nonhazardous materials and harmless reagents.
Ethics
“Science brings society to the next level; ethics keep us there”. – Dr. Hal Simeroth.
Doing science with principles of ethics is the bedrock of scientific activity. The society trusts that the results and the projected outcome of any scientific activity are based on an honest and conscientious attempt by the scientific community. For this reason, we have been thinking about both the societal impact of our project and its compliance with rules & policies.
Responsible Use: ODYSSEE is a triage and not confirmatory test. This basically means that once a person has undertaken it and turns out positive to Tuberculosis, they need to be transmitted to a hospital for clinical exams. We make sure to point that out in our product guide that comes with our kit, and not let the user misunderstand the use of our test.
Product Guide: Our product is supplemented with a detailed and informative product guide in order to ensure the right execution of our test and minimize the risks of human error. In our product guide we also point out the need for a biohazard bin for safe disposal of the test’s components. This way, the release of harmful reagents into the environment is prevented.
Index Card: In terms of privacy, our product guide contains an index card aiming to help healthcare providers match the number of the test they are executing with the right patient. In order to protect the patient’s personal data, we are indicating the use of a barcode instead of the patient’s name. Note: This feature doesn’t fully ensure the patient’s privacy and more measures should be taken.
Accessible Science: As part of a patient-centered care, all patients should receive support from mediators on the field, help them undertake the test. In terms of ending discrimination and encouraging integration, we provide our product guide in different languages. To do that, we first asked from the National Public Health Organization (NPHO) which are the most commonly spoken languages amongst refugees arriving in Greece. Then, we reached out to our fellow foreign iGEMers from this year and asked them to translate our guide.
For Integrated Human Practices purposes, on October 8, we visited the refugee facility "Agia Eleni" managed by the stakeholder INTERSOS. The aim of this visit was to conduct a simulation of our test to verify on-site if it is easy and safe to implement. To perform this demonstration, we made sure it would comply with the rules and policies us they are underlined by iGEM and our University. Before we arranged the visit with the facility's manager, we contacted the iGEM Safety Committee and received affirmation from our University. INTERSOS also confirmed that we executed our simulation following the rules of safety.
