Team:Kyoto/Safety
Safety
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Introduction
Safety was the highest priority in our experiments, and we took every possible measure to ensure that. All
our members attended three lectures about safety: the lecture given by Institute for Frontier Life and
Medical sciences, the lecture about genetically modified organisms (GMOs), and the lecture about chemical
compounds. Throughout those lectures, we understood how to treat chemical compounds, laboratory equipment,
and GMOs. In addition, all experiments were conducted with two or more people to prepare for unexpected
accidents and under the advice of PIs and experts. Just in case, we shared PIs’ contact information and
followed a rule to ask PIs immediately for advice about any trivial concerns.
General organism information
In our project, we used E. coli DH5a, BL21 (DE3), HT115 (DE3), and TOP10. These are not harmful to
humans and belong to risk group 1. We also used Bacillus subtilis which belongs to risk group
1[1][2].
For the "Infection Disease Detection" part, we had to use viruses and a viroid (Dahlia mosaic virus, Tomato spotted wilt virus, Chrysanthemum stunt viroid) that infect plants and are not harmful to humans. In our experiments, these were used only in vitro nucleic acid reactions and were not used to infect plants, so they were not replicated. Moreover, ribonuclease was added after the experiments to avoid the risk of releasing them into the environment. We could not use our lab due to the Covid-19, so we rented part of many labs, which belong to BSL-1 or BSL-2.
For the "Infection Disease Detection" part, we had to use viruses and a viroid (Dahlia mosaic virus, Tomato spotted wilt virus, Chrysanthemum stunt viroid) that infect plants and are not harmful to humans. In our experiments, these were used only in vitro nucleic acid reactions and were not used to infect plants, so they were not replicated. Moreover, ribonuclease was added after the experiments to avoid the risk of releasing them into the environment. We could not use our lab due to the Covid-19, so we rented part of many labs, which belong to BSL-1 or BSL-2.
Prevention of release of organisms into the environment
Before washing each instrument, we fully considered the possibility of contamination by GMOs and got
advice from our PIs as necessary. And before disposal, we autoclaved or sterilized it with hypochlorous
acid or ethanol so as not to release the GMOs to the environment. If Frankliniella
occidentalisescape to the environment, they can seriously damage the floral industry due to the
feeding damage, so we conducted experiments using an icebox under expert guidance, and Petri dishes were
sealed with breathable tape during storage. If we could not find all the Frankliniella occidentalis
visually, we carefully wiped the desk with ethanol just in case. And after all experiments, we drowned the
surviving Frankliniella occidentalis with ethanol.
Use of harmful reagents
In our project, we had to use harmful reagents. According to the safety protocol under the advice of
experts, we used them safely and used the minimum amount necessary. For example, ethidium bromide was used
for nuclear staining, and acrylamide, ammonium peroxydisulfate, and TEMED were used for making
polyacrylamide gel. When we used these reagents, we wore a white coat, gloves, and safety goggles.
Moreover, after using these reagents, we discarded the gloves immediately to reduce the risk of exposing
these harmful reagents. In addition, we used phenol, 2-butanol, and chloroform for gel purification, and
methanol as a fixing solution for tricine SDS-PAGE. After the use of these reagents, we appropriately
treated them according to the advice of our PIs and disposed of them in designated waste containers to
prevent leaking out.
About specific project
Detection: Infection Disease Detection
First, the application contains no cells, so minimize the threat of bacterial contamination.
For practical use, proteins contained in it may be an allergen for some people, and viruses and viroids after detection may be released to the environment. Therefore, it should be strong enough and you need to understand how to use it safely.
For practical use, proteins contained in it may be an allergen for some people, and viruses and viroids after detection may be released to the environment. Therefore, it should be strong enough and you need to understand how to use it safely.
RNAi, Peptide : Killing Pests / Anti-aging reagent / Anti-Bacterial
system
The material used on Frankliniella occidentalis is purified RNA or peptides, so there is no risk of
releasing the GMOs to the environment. However, the safety of the reagents always needs to be considered.
Although these biological materials are expected to be unstable in the environment for practical use, we
need to consider the safety of the users as they may accidentally get these reagents in their eyes or
drink them. Also, when implementing “Killing Pests”, we should consider the impact on the ecosystem.
Firstly, selectively killing Frankliniella occidentalis may affect the balance of the ecosystem. Secondly,
it may unexpectedly kill other insects that have similar sequences to the target sequence of RNAi.
Therefore, we have to carefully consider the implementation, such as conducting tests in a confined area.
Biomolecular Production platform
We had to construct many plasmids and cultivate genetically modified E. coli; therefore all experiments
were conducted following the advice of PIs and experts to ensure that no GMOs were released. For practical
use, this system is expected to be used in a closed system, so the risk of releasing the GMOs is
minimized. In addition, it is considered that E. coli after production can be sufficiently sterilized by
autoclaving. However, there is a possibility that genetically modified E. coli could be released into the
environment. To prepare for that and avoid the impact on plants, additional engineering is required such
as modifying cells to prevent them from multiplying in the environment.
Reference
- Tuberculosis Laboratory Biosafety Manual (Geneva: World Health Organization) (2014), https://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
- IUCN Environmental Law Centre An Explanatory Guide to the Cartagena Protocol on Biosafety, https://portals.iucn.org/library/efiles/documents/eplp-046.pdf