Overview
CAP'siRNA aims to protect sugar beets against Beet Yellows Virus (BYV) infection. Applying purified RNAi precursors encapsulated in inoffensive viral capsids, we prioritize safety to ensure our solution poses no threats to human health, the environment, and biodiversity. Safety is also crucial when carrying out laboratory experiments, using chemicals, and handling GMOs. We have followed the Good Laboratory Practice guidelines set out in European directives to guarantee the safety and security of our laboratories.
Laboratory safety
Laboratory guidelines
The following rules were strictly observed within the laboratory:
Access to the laboratory was restricted and controlled.
Before and after conducting experiments, hands were thoroughly washed.
Wearing a lab coat, closed-toe shoes, and gloves was mandatory for all members.
Eating, drinking, smoking, applying makeup, wearing contact lenses, and wearing open-toe shoes were strictly prohibited.
Each member received proper training from the supervisor before performing any tasks.
Work surfaces were decontaminated after use or in the event of spills or splashes, using 70% ethanol and Phagosoft.
Sharp objects such as needles, scalpels, pipettes, and broken glassware were handled with care to ensure safety and placed in DASRI bins.
Waste management
All waste was disposed of properly to prevent harm to both the environment and human health.
Liquids and contaminated materials that have come into contact with bacteria or biological chassis were decontaminated or inactivated by soaking in bleach for 12 hours.
These materials were never disposed of in regular waste. Instead, they were placed in designated bins for biological or infectious waste.
Project safety
Chemical compounds
During our experimentations, we used several chemicals that present risks. To manipulate, each member was equipped with a lab coat, gloves, security glasses, long pants, and closed shoes. Some substances were also manipulated under a chemical flow hood.
The following table is the list of the hazardous chemicals used by our team during the iGEM competition and the related risks.
| Chemical substances | Related pictograms | Related risks |
|---|---|---|
| Chloroform |
|
Causes skin irritation and serious eye irritation, harmful if swallowed, toxic if inhaled, causes drowsiness or dizziness, suspected of causing cancer. Hazard statements: (H302, H315, H319, H331, H336, H351, H361d, H372) |
| Ethidium Bromide |
  |
Carcinogenic compound (H302, H330, H341) |
| Bleach |
  |
Causes serious skin burns and eye damage. Highly toxic for aquatic organisms. Can create toxic gas if in contact with acidic solutions. Hazard statements: (H315, H318, H400, H411) |
| Ethanol |
  |
Highly flammable, causes skin irritation, serious eye damage. Hazard statements: (H225, H319) |
| Ampicillin |
|
Causes cutaneous allergies, allergic symptoms such as asthma or respiratory difficulties via inhalation (H334) |
| Chloramphenicol |
 |
Causes cutaneous allergies, serious eye damage. Suspected of causing cancer and infertility. Hazard statements: (H318, H351, H361fd) |
| Isopropanol |
  |
Flammable, causes eye irritation, can provoke drowsiness or dizziness. Hazard statements: (H225, H319, H336) |
| TAE 50X |
|
Can cause cutaneous irritation, a cutaneous allergic reaction, and an acute irritation of the eyes. Harmful for aquatic organisms. |
| TEMED |
|
Highly flammable, toxic if swallowed or inhaled, causes severe skin burns and eye damage. Hazard statements: (H225, H302, H314, H331) |
| Sodium dodecyl sulfate (SDS) |
|
Flammable, harmful if swallowed, causes severe skin burns and eye damage. Harmful to aquatic life. |
| Ammonium persulfate |
|
Flammable, can cause allergy or respiratory damage if inhaled, causes skin, eye, and respiratory tract irritation. |
| Monarch® Buffer B2 |
|
Flammable, corrosive to metals, can cause acute toxicity if swallowed, causes skin irritation, eye irritation, respiratory sensitization, may cause drowsiness or dizziness. Hazard statements: (H226, H290, H302, H315, H319, H334, H336) |
| Acetic acid |
|
Flammable, corrosive, can cause skin burns and eye damage. Hazard statements: (H226, H314) |
| Acrylamide/bis-acrylamide |
|
Harmful if swallowed or if inhaled, can cause skin irritation, may cause genetic defects, allergic reactions, cancers, damage to organs with repeated exposure, suspected of damaging fertility. Hazard statements: (H302+H332, H315, H317, H319, H340, H350, H361f, H372) |
| Coomassie Brilliant Blue |
|
Highly flammable, toxic if inhaled, can cause skin and eye irritation, damage to organs. Hazard statements: (H227) |
| Hydrochloric acid (fuming) |
|
Corrosive to metals, causes severe skin burns and eye damage, may cause respiratory irritation. Hazard statements: (H290, H314, H335) |
| TRIzol |
|
Causes severe skin burns and eye damage, can cause damage if swallowed or inhaled, may cause respiratory irritation and damage to organs, suspected of causing genetic diseases. Hazard statements: (H301, H311, H314, H318, H335, H373, H341) |
| EDTA |
|
Toxic for aquatic organisms, hardly biodegradable, can cause irritation. Hazard statements: (H319) |
Organisms used
- Escherichia coli strain DH5-alpha:
production of DNA bank. This non-pathogenic strain does not produce any compound harmful to human health [2].
- Escherichia coli strain HT-115:
genetically modified to produce RNAi precursors. This strain is non-pathogenic, and the genetic modifications do not change the severity of the bacteria towards human health [2].
- Escherichia coli strain BL21:
genetically modified to produce the capsid protein of the TMV. This strain is also non-pathogenic, and the genetic modifications do not change its severity towards human health [2].
- Tobacco Mosaic Virus (TMV):
Tobamovirus, TMV-U1.
- Beet Yellows Virus (BYV):
Closterovirus, BYV-BBRO.
The TMV and BYV are nonpathogenic viruses for humans and can be handled in a Level 1 laboratory security level [3]. They are widely present in the environment and the BYV is transmitted only by aphids. French public research institutes have vetted them both.
Myzus persicae: used for the transmission of BYV. The aphids that we have do not possess wings so they cannot spread, are widely present in the environment, and were validated by the iGEM safety comitee.
Product safety
The dry lab team created a tool to ensure the safety of our product, mainly of the long hairpin RNAs (lhRNAs) present in it. This tool, called, SafeRNA, was designed to ensure the specificity of our lhRNAs. To do so, it predicts, every small interfering RNAs (siRNAs) that could be formed from our lhRNA and verify that they do not target humans, sugar beets, or their microbiome. Overall, it showed that our lhRNA do not have any relevant off-target on the organisms that were tested (see more details on the Software part). Moreover, the capsids encapsulating the lhRNAs are non-GMO, non-pathogenic and widely present in the environment under the form of the wild type TMV. No danger could come from it. Therefore, the assembled solution causes no risk to either human health nor biodiversity in general.
References
[1] European Chemicals Agency. Pictogrammes CLP (2024). Available at: https://echa.europa.eu/fr/regulations/clp/clp-pictograms (accessed on the 09/23/2024)
[2] Virginia Tech. E. coli Strain Information. Research and Innovation (2024). Available at: https://www.research.vt.edu/ibc/protocol-applications-and-submissions/e-coli-strain-information.html (accessed on the 09/23/2024)
[3] Zhang, L., Wu, C., Yang, Y., & Zhan, X. The effects of water temperature and salinity on the growth and survival of larval Penaeus vannamei. PLOS ONE, 8(4), e60621 (2013). https://doi.org/10.1371/journal.pone.0060621
