Our project METIS is an in vitro diagnostic device that is able to detect certain microRNAs that are deregulated in people in the early stages of Pancreatic Ductal Adenocarcinoma (PDAC). Specifically, we aim to detect and quantify these molecules in order to provide a safe conclusion on whether or not someone is in the early stages of PDAC. So far, this is not possible with the current available diagnostic techniques. Our goal is to improve the prognosis of the disease, by detecting cancer when it is still localized, so that the patient can succeed in overcoming the fatal risks. We have managed to design a toolkit that poses no major threat to the environment or people. Nevertheless, we are completely aware of the importance of safety measures that need to be taken in order to ensure that even the slightest chance of risk will be minimised. Therefore, we are providing you, in the following segments, with the detailed safety measures taken by our team.
Biosafety - Regulations
As mentioned in previous pages, our project involves the design of an in vitro diagnostic tool whose operation is based on synthesized RNA molecules called toehold switches. More information on the function of these molecules can be found in the "Project Description" section. Toehold switches functionality does not require the presence of bacteria or any other microorganism. Nevertheless, the use of a chassis organism is necessary for the contriving and their experimental control. However, this fact gives us the opportunity to select the most harmless microorganisms available. Thus, for the development of our project we chose these three E.coli strains:
- ~ DH5a
- ~ DH10b
- ~ BL21
In the documents listed in the links below, it is clearly stated that these particular strains, which are classified as risk group 1, are unlikely to give rise to safety issues concerning human and environmental health. Specifically, in the WHO Laboratory Biosafety Manual and the directive 2000/54/EC of the European Union on the protection of workers from risks related to exposure to biological agents at work , this affirmation can be found.
Since our laboratory is classified as Biosafety Level 1 , we are in position to experiment with Risk group 1 microorganisms. The risk of these microorganisms to harm the health of humans and cause environmental problems is insignificant. The confirmation of the above can, also, be ascertained in the Official Government Gazette of the Hellenic Republic along with the European Union Directive 2009/41/EC for the contained use of Genetically Modified Microorganisms.
You can find these documents in the links below:
We also made sure that we designed our protocols to meet the needs of the Safety Policies guidelines mentioned above in order to ensure that we avoided any unnecessary harm to the wet lab members, environment or society.
Below we provide you with the completed safety form of the iGEM Committee:
In order to reduce the risks associated with our project and experimental procedures, we consulted our Principal Investigator and Advisors, from the Aristotle University of Thessaloniki, when designing an experiment. In addition, we have received a training workshop by Dr. Papadopoulou Lefkothea, in order to conform to the Basic Lab Biosafety Rules and Regulations, focused on the risks arising from the experiments that we are planning to perform.
We were informed about the laboratory equipment and how it must be properly used and cleaned after the experiments as well as how we should act in case of emergency. Some basic rules that we were obligated to follow during this time were to always use protective coats, one-use disposable gloves and protective masks due to COVID-19 measures. Special attention was paid when using the UV- light. We were instructed to wear protective UV-glasses and a protective screen. During this training, we were also informed on the waste management protocols of our institution, as well as for the handling of hazardous substances used and how to report an accident to the supervisor of our lab.
Our lab was fully equipped with the essential technical devices that we needed in order to complete our project and also there were MSC and PhD students who were ready to help and inform us on anything we needed.
In addition to the above, we took the initiative to create a list of rules concerning the way we operate in the laboratory according to the experience and scientific expertise of Dr. Papadopoulou. The purpose of this initiative is to provide a handbook of advice to future iGEM teams to which they can refer to and consult at any time. Furthermore, when they obtain the necessary experience during their time experimenting in the lab they will be able to complete and improve this list for the upcoming team. Thus, we will ensure that over time a complete guide will be established, complemented by many perspectives, which can be used not only by iGEM teams but also by students of each school in their respective laboratories. In this way, we will secure safety for lab members, their associates, the environment, and society as a whole.
You can find this document by clicking the button below:
Laboratory Manual on Safety Guidelines
You can find this document by clicking the button below:
Laboratory Manual on Safety Guidelines
| What if a false - positive testing occurs?A false positive result implies that someone without PDAC will be diagnosed as a patient.
This may trigger a series of diagnostic tests that could potentially be invasive or not. Either way, this unnecessary hardship may affect one’s health and psychology in general. Apart from blood samples, testing may include ultrasound , MRI or PET/CT- scan which can be harmful due to radiation. Of course, we can not fail to mention the impact that such a diagnosis will have on the patient and his/hers family environment. A sphere of concern will be created unnecessarily due to misdiagnosis which will probably justifiably lead to anxiety and distress. Treatment also may cause side-effects. We must also emphasize the significant financial impact on the “patient’’. Regarding the treatment of these diseases, the patient will be subjected to a huge futile expense due to the exorbitant cost of medical care and medicines .
| What if a false - negative testing occurs?A false - negative result implies that someone with PDAC will be diagnosed as a healthy individual.
When a false-negative test occurs a very serious issue arises. The patient may think he/she is healthy but in fact suffers from PDAC without being aware of it. Of course the disease may be in its early stages but the fact that the patient does not know about it is life - threatening. A potential progression may occur which could lead to an irreversible condition.
| Our solutionIn order to counter these problems we have come up with some solutions. Firstly, we definitely need to make our tool more reliable in order to minimize the case of false-testing. To do that, measuring the concentration of the three micro-RNAs we will be using is obligatory. Our aim is to establish the specificity and sensitivity percentage of the kit. The only way this can be achievable is by putting our device through clinical trials in PDAC patients. Protocols will be followed along with the procedure ensuring that patients consent to the use of their biological samples and that their information will be kept safe.
One way to limit false testing is to suggest our diagnostic tool as a simple, quick and easy-to-do test that will give us a fast screening. After some time a second test could be performed in order to ensure more reliable results. Best results of usual rapid tests are ensured through repeatability, so we could take advantage of this fact. In general, people over the age of 60 should have this test but the results should be discussed with their physician who will decide whether or not more tests are necessary in order to come to a safe conclusion about the diagnosis. We suggest our kit be used as a first-line rapid test which will be followed up by the best suited imaging technique according to the specialist.
Our team intends to collaborate with specialists in the field of biotechnology to further improve our device making it most reliable. In all cases, our kit will contain the necessary calibrators and negative and positive controls to ensure that there will be no noise from the device in the final result.
| ConclusionNonetheless, our team aims to improve the device and reduce the risks mentioned above to a minimum. Besides this, our project does not induce any genetic alteration or interferes in any way in human physiology, psychology, and overall health. Our goal is to provide the world with a safe, fast and reliable test so that it can act as a complementary testing to the valid and early diagnosis of PDAC. Today the early diagnosis of this devastating disease is insufficient and when it is detected in the late stages it is no longer manageable. Therefore, we want to contribute positively to the reduction of this -diagnostic- gap and to science in general. In this way, we will be able to ensure the early treatment of the disease so that the patient has an effective recovery and a better quality of life.
Our team recognizes that despite any technological advances currently happening, it is absolutely important to protect the environment and maintain a balance in natural habitats. On this page we managed to list the risks that can potentially cause environmental damage or threaten human health.
An important risk associated with our experimental procedures, is the antimicrobial resistance genes that will be used as selection markers. A potential hazard may occur if the genetically modified organisms containing these genes happen to escape into the environment. A significant effect of that would be the spread of antimicrobial resistance to other wild type bacteria species existing, though horizontal transfer. So eventually a formation of pathogens resistant to medicine could occur.
| Possible hazards during our experiments
In our laboratory we experimented with commonly used antibiotics such as chloramphenicol, ampicillin, and kanamycin as well as their resistance genes. Many human infections are treated with use of these antibiotics and are also listed by WHO as Critically Important Antimicrobials. Hence, we consider that one of our top priorities is to preserve the efficiency of the antibiotics mentioned above by making sure that none modified microorganism will escape to the environment.
Although our project is not fully developed, we considered the possible hazards during its distribution and usage.
| Possible hazards during production and distribution
As mentioned in the previous safety pages, for our project, the engineered microorganisms are only used as a chassis. This means they are used for keeping and amplifying the toehold switches we designed and they are not a part of the diagnostic system. This means that an escape to the environment is unlikely. However, metabolism products or parts from these bacteria could potentially escape.
As far as the reagents are concerned, they can be divided into two categories: the DNA sequences, which include the toehold switches and the chemical reagents, which consist of the cell-free system reagents. For both categories, there could be a small chance of escape during their distribution. However, the DNA sequences do not pose any risk to the environment and therefore, cannot be considered contaminating or hazardous. The chemical reagents of the cell free system, according to the manufacturer, are not considered hazardous. However, as chemical substances, they should always be handled with caution and according to the safety data sheets.
Finally, there is another important risk concerning the safety of the end-user. We expect our method to be performed by the personnel of a clinic or a diagnostic center. These employees will be handling urine samples and as a result they could be exposed to any infectious agents contained in the urine of the patients. Although the personnel will be trained to handle biological samples, it is an important risk that we took into consideration.
To reduce the risks mentioned above, in its final form the kit will come with a board - on which 2 separate reactions will take place - as well as the necessary reagents. The purpose of performing these reactions is to quantify certain microRNAs in the urine of patients when inserted in the device. Firstly, we need to ensure that the quantity of these molecules is sufficient enough to be detected. For that reason, an amplification reaction occurs and is followed up by the binding of the selected miRNAs onto toehold switches. The result of this phenomenon is the unfolding of the hairpin which induces the expression of eGFP protein.
- Additionally, in case our product is mass produced we must pay more attention to it . The company that will produce the toehold switches and the kit as a whole, should operate under studied protocols and in specialized laboratories so that the bacterial strains will not escape into the environment and contaminate the end-product.
Another important factor to consider is making our product completely recyclable. So, for that cause the materials need to be chosen carefully. Our goal is delivering an environmentally friendly as well as completely safe to the end-user diagnostic test. An issue that arises from the wide use of the product is its disposal. Along with the disposal of the product we need to take into account the urine of patients that has been examined and centrifuged. Special bins need to be established in the laboratories - especially during clinical trials- to make sure that end-products won’t be able to contaminate any reagents or the lab staff.