Overview
One of the things we value and care about the most in our team is the safety of our team members and our community, throughout the entirety of our experimental procedures. As a result, we made it a priority from the start to find a secure workspace that adhered to E.U. safety measures and guidelines and to ensure thorough safety training for all of our members. In addition, we aimed to include non-pathogenic organisms in our project to reduce the risk of hazardous exposure to the team and the environment.
Lab Safety Measures
Our working environment is a standard microbiological lab and is classified as biosafety level 1 (BSL-1). For the conduction of the experiments, we used open bench areas, a specialized greenhouse, chemical fume cabinets for dangerous chemicals, and a laminar flow cabinet to prevent contamination of our microbiological cultures. Our project does not involve any prohibited activities, and all of the organisms and parts used are on the competition's White List, according to iGEM regulations.
Living organisms and Parts
Our project involves the creation of a novel diagnostic tool utilizing Leucine Rich Repeats(LRRs) for the diagnosis of plant pathogen viruses of the genus tospovirus before symptoms appear. We used an iGEM part, OmpA BBa_K1489002, to surface display the LRR and NB-LRR domains of Sw-5b resistance gene of Solanum peruvianum on E. coli DH10B cells. A small region of the movement protein of TSWV (Nsm115-135) is required for NLR recognition. The movement protein normally contributes to pathogenicity, but in absence of other viral proteins and viral genome, there is no danger of infection and transmission. The movement protein was C-terminally fused with YFP to be traceable when bound to the surface-displayed binder.
As part of our experimental procedures, we used living microorganisms, such as E. coli DH10B and tumefaciens C58C1 cultures, to express and multiply our vectors. We also agro-infiltrated two plant species, N. Benthamiana and N. Sylvestris to ensure the success of our experiments.
Project risks
Mutagens, highly flammable chemicals, and other hazardous chemicals are used in our project such as Ethidium Bromide and Absolute Ethanol. Using such substances may potentially lead to human health risks if mistreated, yet by following the safety protocols to the letter, this danger is nullified. Furthermore, UV light could be mutagenic, thus protective gear was used and exposure was minimized. Ethidium Bromide was used exclusively in a laminar airflow cabinet as it is volatile and was handled with special gloves. Sterile conditions were established by careful use of flame. Also, none of the chemicals were inhaled or ingested on purpose.
Managing risks
University faculty including professors and post-doctoral researchers as well as the Bioethics Board of our institution were advising us along our experimental endeavors and eliminated the risks, while also ensuring the smooth conduction of the protocols. In addition, safer practices were proposed by our PIs in order to avoid any misfortunate incidents, such as the usage of laminar flow instead of the flame for sterile conditions.
On our part, we received safety and security training from specialized staff to avoid any dangerous mishandling of the laboratory equipment and reagents. We were informed, among others, about lab access and rules, differences between biosafety levels, biosafety equipment, good microbial technique, disinfection and sterilization, emergency procedures, rules for transporting samples between labs or shipping between institutions, chemical, fire, and electrical safety.
We decided against using entire living plant pathogens as the risks involved would be disproportional to the anticipated rate of amelioration of our Project.
Our institution, as well as the host lab, have taken all the necessary measures to satisfy the guidelines set by the E.U (GLP Good Laboratory Practice OECD), the Greek government, the Ministry of Research, and the host institutions(University of Crete and Foundation for Research and Technology).
Those guidelines include but are not limited to, special training provided to all team members regardless if they will perform lab work or not, a thorough explanation of the usage guidelines of all lab equipment as well as meetings aimed at clarifying any "dark spots" in the protocols that will be followed. Moreover, a line of communication has been established with the Biosafety and Bioethics Boards of our institution, if need be.
Future applications
In the case that our LRR-based diagnostic tool is fully developed and available for usage, it could be beneficial in an industrial environment, in agricultural facilities, but also a form of a consumer product or a small enclosed device as a rapid test, bio-sensing strip with cells that detect tospoviruses.
If LRR-based diagnostic tools prove to be equal to or better than existing ones utilizing antibodies, the limits are endless when it comes to potential targets that can be diagnosed both in plants and in animals.
The diagnostic tool could be used in agriculture, ergo in the field. In that case, a sample would be isolated from a plant and the test would take place on the spot. For that, specific forms need to be filled in with our institutions according to the standards set by the Greek government, to use a product of synthetic biology.
Although there could be a risk of harm to agricultural animals, crops, or domesticated animals and the environment, including wild plants and animals, our team did not identify any bad outcomes for future uses.
Our final product (diagnostic tool) does not involve any engineered organisms, thus no risk of spreading, while the creation of certain substances in the laboratory may involve engineered organisms. The latter though will take place in a highly controlled environment.