Agriculture of any kind affects the environment, and the use of new genetic techniques in agriculture is no exception. In this context, our project Agrocapsi, aims to develop a sustainable, non-toxic fungicide that prevents the wilting of chili crops.
This project makes use of genetically modified organisms by inserting heterologous genes for protein production. Therefore, strict bio-containment measures are required to effectively prevent the unwanted release of the modified bacteria into the environment. The effects of our modifications were meticulously scrutinized.
All team members completed the courses listed below, provided by the Public Health Agency of Canada web page, being successfully certified by them:
Autoclaves
Biological Safety Cabinets
Biosafety in the Classroom: Post-Secondary Laboratories Course
Biosecurity Plan
Conducting a Biosecurity Risk Assessment
Containment level 1: Physical Design and Operational Practices
Ensuring everyone's safety inside and outside the team, certain basic lab rules were always followed, avoiding common risks inside a laboratory.
Personal Protective Equipment
We always used the Personal Protective Equipment (PPE) for safe practices.
When leaving the lab we removed the PPE because it shouldn't be used outside the lab unless it's packed and well sealed. Disposable materials and consumables were properly discarded, while the other reagents and materials were tidily arranged into place.
Hygiene Measures
We washed our hands when entering and leaving the lab. We also disinfected them with 70% ethanol before and after putting on the gloves.
Safe spaces
We left our personal belongings away from the lab to avoid any cross-contamination in both ways.
| Autoclaves | Biosafety Cabinets | Burners | |
|---|---|---|---|
Autoclaves work to achieve steam sterilization through high temperatures and pressures. This allowed us to work with microbial free instruments and containers. Our lab has 3 of them:
|
Biosafety cabinets maintain a sterile environment to prevent contamination of the material we are using. They avoid that the observer or external agents contaminate the sample as well as being a containment barrier that keeps the sample from contaminating our surroundings. Our protocols included the disinfection of the cabinet with 70% ethanol and UV light for 15 min before its use. Our hands and any material that entered the cabinet had to be sprayed thoroughly with 70% ethanol. When done using the biosafety cabinets, we cleaned them with 70% ethanol and let the UV light run for another 15 min. | We use Bunsen burners to create a sterile halo when working on an open bench. First we disinfected the working area with 70% ethanol, later placing 2 burners with a maximum gap between them of 20 cm, increasing the sterile working area available. | |
| Bacteria incinerator | |||
| We used a bacteria incinerator to sterilize the inoculation loops. This applied when we wanted to inoculate inside the biosafety cabinet. | |||
Access
The labs we worked in are Containment Level 2 Laboratories. These have lockable doors and restricted access to biotechnology alumni and faculty teachers. Students must complete a safety course before entering any of the laboratories.
Waste Disposal
Laboratory waste is usually classified into four types depending on their nature. Each of them should be discarded accordingly to avoid accidents or contamination. Non-solid biological wastes go in a yellow hermetic bottle, while the solid ones go in a yellow bag. Sharp objects belong in a thick red plastic bottle. Materials that were in contact with bacteria or any kind of infectious agent should be disposed in a red bag. Due to the characteristics of the project, we needed to work with bacterias, therefore using mostly red containers (bags and bottles accordingly).
Choosing a non-pathogenic chassis
Our team is all into safe laboratory practices. We want to cut the risk of the team, community and environment. This is why we chose a non-pathogenic chassis. We worked with two strains of Escherichia coli: BL21 (DE3) as the expression system for the AMPs and HT115 for the iRNA synthesis. These strains are categorized as Risk Group 1 organisms, meaning they don't generally cause disease nor represent an environmental risk.
Despite their low risk level, all protocols involving these organisms took place in a biosafety cabinet while wearing PPE.
Working with safe parts
The parts we used for the development of our project come from:
iGEM's Registry
Protein coding genes from plants
None of these are toxic to humans, animals or the environment in any way.
Phytophthora capsici
We worked directly with Phytophthora capsici and Fussarium solani. The oomycete, P. capsici, provokes wilting a disease that can destroy up to 100% of chili crops. Thus the importance of working with it. Fusarium is a beneficial fungus and it's important for us to analyze if our product affects it. These two microorganisms fall into the Risk Group 1 category which makes them low risk to work with.
The following practices of biosafety and biocontainment were done to ensure the proper management of the fungus:
We worked with these fungi under sterile conditions, designating an exclusive incubator for their storage, helping minimize the contamination risks.
