Safety

Overview

In 2024, the WUXI-DSAS iGEM Team aims to develop an eco-friendly, non-toxic biological fungicide designed to protect corn from sheath blight. Ensuring the safety and harmlessness of our product for both humans and the environment is a priority. To achieve this, we have focused on three key areas: laboratory safety, project safety, and product safety. Below are the safety measures we have implemented.

Laboratory Safety

Our experiments are conducted in the iGEM Laboratory at Zhejiang University, which is equipped with essential safety facilities, including eyewash stations, sprayers, a first aid kit, and fire extinguishers.

Laboratory Safety rules:

1. Students who enter the laboratory to engage in teaching and scientific research activities must strictly abide by the rules and regulations of the laboratory.
2. Safety education must be received before the experiment. Carefully preview and prepare for the experiment as required.
3. Wearing lab coats, masks, and gloves when entering the laboratory.
4. No skin should be exposed in the lab environment.
5. Keep quiet during the experiment, do not make loud noise, do not smoke.
6. Eating and drinking are prohibited in the laboratory to minimize the risk of contamination and keep a clean, controlled environment.
7. Take good care of instruments and equipment and save materials.
8. Once an accident occurs in the laboratory, instructors should be connected in time and properly handled. All kinds of experimental wastes should be classified and disposed according to the requirements of the laboratory. Pipettes, spear heads and slides used in etiological experiments should be put into the disinfection tank in time for disinfection, and should not be placed in other places or washed into the sink to prevent contamination.
9. Before operation, carefully read the experimental instructions or instructions, operate strictly according to the operation requirements, and do not operate the instrument blindly.
10. The temperature, voltage and current of the water bath box, incubator and electrophoresis apparatus in the laboratory have been adjusted before the experiment. No one is allowed to turn any knob of the relevant instruments in the laboratory, so as not to affect the normal operation of the experiment.
11. When using the centrifuge, the centrifuged sample must be balanced, and the speed of rotation must be observed to prevent the centrifuge tube from breaking. The centrifuge cover must not be opened until the centrifuge has come to a complete stop to ensure safety.
12. Before using electrical and electronic instruments, check the power supply and external wiring, and then turn on the power switch. Turn off the power switch of the instrument in sequence after the experiment. It is forbidden to turn on and off the power switch frequently.
13. Do not use excessive force when turning the knob to avoid damaging the instrument. Take care not to drop or splash the liquid on the instrument.

Project Safety

Our biological fungicide, designed to protect corn from sheath blight, will be applied in actual cornfields. Given that these fields are outdoors, it is crucial that our fungicide is safe for the environment. To ensure this, we have taken several precautionary steps.

Organisms

We selected E. coli HT115 (DE3) as the vector for expressing dsRNA due to its RNase III-deficient nature, which preserves dsRNA integrity, making it ideal for large-scale dsRNA production. To test the fungicide’s effectiveness and safety, we used Rhizoctonia Solani, the fungus responsible for sheath blight. During experiments, team members wore lab coats, gloves, and masks with no skin exposure, and sterilization was performed after each experiment to ensure safety.

dsRNA Extraction

We evaluated four methods for dsRNA extraction: chloroform reagent, Trizol reagent, 75% concentration alcohol precipitation, and RNA-easy isolation reagent. We chose the latter two methods because they are safer for both humans and the environment, avoiding the use of chloroform and Trizol, which are toxic and can cause environmental contamination.

RNAi Therapy

The RNA interference (RNAi) can target the target genes of pathogens, the double-strand RNA molecules can effectively trigger RNAi silencing of specific genes. Based on the RNAi technique, we used SIGS (spray-induced gene silencing). The target double-strand RNA (dsRNA) was artificially synthesized and applied to the superficies of fruits and vegetables, concurrently associated with pathogens. Consequently, the pathogens were deterred and immobilized via the cross-border conveyance of dsRNA and the inhibition of target gene transcription. This technique is straightforward to execute and independent of the host genetic transformation system. The core constituent of this method is the biological macromolecule dsRNA, which is innocuous and leaves no residue. For dsRNA molecules, safety can be ensured by the following aspects.

Environmental Friendliness

Our nucleic acid-based fungicide is biodegradable in natural conditions, unlike chemical fungicides that leave harmful residues. RNase, an enzyme that degrades RNA, is widely present in the environment, ensuring that our product does not persist and harm the ecosystem. Additionally, our fungicide is selectively toxic to the target fungus without affecting other organisms.

Food Safety

Corn is a crucial food source for both humans and animals, and sheath blight significantly reduces corn yields. Our product effectively lowers the production of catalase in Rhizoctonia Solani, protecting corn and ensuring a safe food supply.

Non-GMO Product

Unlike genetically modified organisms (GMOs) that alter plant genetics, our product does not involve genetic modification, thus avoiding potential risks associated with GMOs. Instead of killing fungi, our product silences the CAT gene in Rhizoctonia Solani, preserving the food chain and indirectly protecting other species dependent on the fungus.

Product Safety

Our fungicide is derived from dsRNA expressed by Escherichia coli and targets the CAT gene in Rhizoctonia Solani, reducing harmful hydrogen peroxide production. This makes it a safe defense mechanism for corn plants against fungal infection. Our product is non-toxic to humans, degrades naturally in the environment, and poses no threat even if ingested, offering a safer alternative to conventional pesticides.

To address public concerns, we interviewed corn farmers, who highlighted safety as their primary concern about crop chemicals. Our product addresses these concerns by providing an effective, non-toxic alternative to traditional chemical treatments, ensuring the safety of both the environment and food supply.