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Overview

To ensure that our project is safe and harmless to both humans and the environment, we have made significant efforts in three aspects: laboratory safety, project safety, and product safety. The following are the safety measures and precautions we have considered for this iGEM project.

Lab Safety

Lab configuration

Our experiments are conducted in the iGEM laboratory at Shenzhen University. The laboratory is equipped with a laminar flow hood, a fume hood, a first aid kit, fire extinguishers, waste liquid containers, and other necessary safety equipment. Additionally, there are nearby shower facilities, eyewash stations, and firefighting facilities.

Figure 1. Our fire extinguishing equipment.

Safety training required by school

As biology students, it is important for us to prioritize laboratory safety. In our freshman year, we are required to undergo laboratory safety training. This includes studying and memorizing the laboratory safety manual, as well as participating in a laboratory safety exam. To earn the corresponding credits, we need to achieve a score of 90 or above in the exam. These measures ensure that we have a solid understanding of laboratory safety protocols and are equipped with the necessary knowledge to conduct experiments safely.

Wet Lab Training

Before conducting iGEM experiments, we had the opportunity to visit the laboratory under the guidance of senior students. During the visit, we familiarized ourselves with the laboratory equipment and learned how to use them properly. Additionally, we had the privilege of watching videos recorded by previous iGEM participants, which provided us with valuable insights and practical knowledge related to the competition. These experiences helped us gain a better understanding of the laboratory environment and the specific requirements of iGEM projects.

Figure 2. Image from a previous iGEM laboratory tutorial video.

Laboratory Specifications

Before entering the laboratory, it is mandatory for us to follow certain safety protocols. These include wearing laboratory coats, masks, and gloves. We are not permitted to wear open-toed shoes, and our hair must be tied up. Eating and drinking are strictly prohibited within the laboratory premises. These measures are in place to minimize potential contamination risks, ensure personal safety, and maintain a clean and controlled work environment.

Figure 3. Our iGEM team member is conducting experiment.

Project Safety

Our project includes two parts: RNAi biological pesticide and plant immune preparation. which will be packaged into our product PolycoBead, which will be finally applied to farmland after the production and optimization of the process. Farmland is an open environment, which means that our products will inevitably come into contact with the outside world. Therefore, we must ensure the safety of the product in the whole process from production to application.

Organisms

We selected two commonly used strains of E. coli BL21 (DE3), and E. coli HT115 (DE3) as the vectors for protein and RNA expression in the laboratory. In product application, we used Bacillus subtilis WB800, which is used as commercial biological control agent that has been widely promoted. Also, it has been used by multiple iGEM teams, confirming its safety. In tomato infection, we selected a very common strain of Botrytis cinerea, which is frequently found on decaying food.

To ensure their safety, we performed the following two tasks, checking them in the iGEM white list and verifying if they are prohibited for use as infectious microorganisms in the Catalogue of Human Pathogenic Microorganisms for Infectious Diseases Control. The strains using in the laboratory, E. coli BL21 (DE3), Bacillus subtilis WB800, E. coli HT115 (DE3), and Botrytis cinerea, are listed on the iGEM white list. This helps maintain the focus on safe and responsible genetic engineering practices throughout the iGEM community.

To ensure their safety, we performed the following two tasks, checking them in the iGEM white list and verifying if they are prohibited for use as infectious microorganisms in the Catalogue of Human Pathogenic Microorganisms for Infectious Diseases Control. The strains using in the laboratory, E. coli BL21 (DE3), Bacillus subtilis WB800, E. coli HT115 (DE3), and Botrytis cinerea, are listed on the iGEM white list. This helps maintain the focus on safe and responsible genetic engineering practices throughout the iGEM community.

Figure 4. Our strains are not on the list.

RNAi therapy

Based on RNAi technology, RNAi-mediated biopesticides can target target genes of pathogens and produce silencing effect. We used E. coli HT115(DE3) to produce our shRNA molecules and used them as our RNAi biopesticides strain in the laboratory and has been widely used in the industrial fermentation of biomolecules, so it is safe in the production process. Cell-penetrating peptides are biomolecules that are released into the environment and are able to degrade after a period of time without residue. For shRNA molecules, safety can be ensured from the following aspects:

• 1. Specific targeting
  In order to ensure the species specificity and biological safety of our shRNA, after selecting the target of the pathogen, The CDS sequence of this target in B. cinerea was input into the total nucleic acid database of the National Center for Biotechnology Information (NCBI) website for BLAST to query the similarity of neighboring species homology. At the same time, the target position of the designed siRNA sequence was guaranteed to be the non-conserved region of the sequence. Then, we entered the candidate siRNA fragments into the total mRNA database for BLAST, and the siRNAs with 80% match to genes in other common species (such as human, tomato, dog, rice, wheat, etc.) were excluded, so as to ensure the sequence specificity.



Figure 5. BLAST results of inputting the target gene's CDS sequence into the nucleotide database.


• 2. Environmental friendliness
  RNA molecules are easily degraded by nucleases distributed in the air. Even when coated with CPP, CPP-shrna will not always exist in plants or soil after playing its role, greatly reducing the possibility of targeting other species and organisms. Unlike chemical pesticides, which remain in the soil, RNAi biopesticides are environmentally friendly.


• 3. Controllability
  At present, RNAi is mainly used in crop protection through two ways: host induced gene silencing (HIGS) and spray induced gene silencing (SIGS). To adopt HIGS means to transform crops through transgenic means, however, people's acceptance of Genetically Modified products is still not high in China, so this method can not be well applied in real life. We soon noticed SIGS, which is an emerging, non transgenic RNAi strategy. Under the condition that pathogenic fungi can absorb RNAi molecules from outside, RNAi molecules can also play a good role in cross-kingdom silencing. Through SIGS, RNAi biopesticides can cause no permanent changes in plant gene expression and they are controllable.

Engineered B. subtilis

• 1. Selection of chasisg
  Our project expect to be able to use a bacterium that can colonize the plant rhizosphere to continuously release immune factors to prompt the plant to mount an immune response. B. subtilis is one of the most widely accepted plant probiotics and is naturally existing in soil. It has been reported that B. subtilis have control effects on different fungal diseases in different plants, such as rice rhabdomyosis and wheat take-all in cereal crops, tomato gray mold and apple mildew in vegetable and fruit crops, and they have good control effects on a variety of crops and fungal diseases. The use ofB. subtilis ensures that our chassis can function well in the soil without harming the balance of the soil microbial community and causing harm to the plants.


• 2. Expression of BvEP
  To trigger the immune response in plants, protein phosphomutase BvEP from Bacillus velezensis LJ02, which is homologous to B. subtilis was used as an immune-inducing factor. It has been shown to trigger ROS outbreak and the expression of related enzymes by inducing PTI and ETI immune pathways in tomato fruits. At the same time, it has been verified that the application of this element on tomato has no significant effect on the weight of tomato fruit and the content of various nutrients.


• 3. Sucrose-suicide switch
  To prevent the survival of our engineered bacteria from flooding the croplands, we designed a sucrose-induced toxin/antitoxin based system as a suicide switch, which includes the mazE and mazF genes. The mazF gene encodes the toxin protein MazF, which in its own presence causes cell death. In the presence of sucrose, the mazE gene encodes the antitoxin protein MazE, which binds to MazF, rendering it ineffective. When sucrose and engineered bacteria were mixed and sprayed into the field, sucrose was slowly lost after falling to the field, and the inhibitory effect of sucrose on MazF was gradually reduced. When sucrose was depleted, the engineered bacteria stopped expressing the MazE protein and encoded the toxin MazF, turning on the suicide mechanism.

Figure 6. Our suicide switch.

To learn more, please click Design.

Product Safety

PolycoBead

In order to guide agricultural related personnel to use our products and regulate the dosage of pesticides, we designed the product PolycoBead, which encapsules RNAi-based biopesticides and plant immune inducing perparation whicn forms a bead as the outer film to wrap our CPP-shRNAs preparation and the engineered chassis-bacteria B. subtilis together to form a bead. In addition to the main ingredients, we have also fully considered the safety of PolycoBead packaging materials and accessories in the design of their safety.

• 1. PolycoBead features an outer membrane composed of highly hydrolyzable water-soluble polyvinyl alcohol (PVA) film. PVA film offers excellent biocompatibility and biodegradability, being the only vinyl polymer that can be utilized as a carbon source by bacteria. It naturally decomposes through microbial and enzymatic action, degrading by 75% within 46 days, ultimately breaking down into CO2 and H2O. Extensive biological testing has confirmed its non-toxicity to living organisms. Due to its water solubility and biodegradable properties, the liquid formed when PVA film dissolves in water and infiltrates the soil can enhance soil aggregation, aeration, and water retention. This is advantageous for plant growth and survival.


• 2. We have utilized glycerol (also known as glycerin) to provide the liquid environment within the PolycoBead. Glycerol is a commonly used polar solvent for preserving biological samples. Although trace amounts of glycerol-water mixture may enter the soil, it is also biodegradable by soil microbial communities.


• 3. In order to minimize interference from the engineered bacteria on the CPP-shRNAs complex and ensure the viability of Bacillus subtilis, we employed the method of calcium alginate encapsulation. The use of calcium alginate encapsulation for immobilizing cells is a widely adopted technique known for its high encapsulation efficiency and minimal toxicity to microorganisms. Additionally, when the calcium alginate-engineered bacteria complex is introduced into the environment, the gradual loss of calcium ions results in the dissolution of the calcium alginate matrix, facilitating the release of the bacteria. Through experimental validation, we have demonstrated that a single calcium alginate bead can completely release its contents within 40 hours. The residual components are non-hazardous to soil and gradually degrade over time.

Use of pesticides

We visited Guangdong Hongke Testing Technology Co., Ltd., in order to consult specifically about the safety issues related to pesticide testing. This visit to a professional testing organization was aimed at gaining a deeper understanding of the safety aspects concerning agricultural products.

Figure 7. Our visit to Guangdong Hongke Testing Technology Co., Ltd.

We visited the Pesticide Residue Testing Center at the Agricultural Bureau to gain insights into the safety issues related to pesticides. Director Yin emphasized the strict requirements and procedures for pesticide sales. Additionally, he highlighted the importance of developing a agricultural product testing plan to ensure the quality and safety of agricultural products. This plan involves determining the scope, frequency, and targets of the testing to ensure compliance with standards. These measures contribute to safeguarding the quality of agricultural products and protecting consumer health.

Figure 8. Our visit to the Pesticide Residue Testing Center.

Director Chen Wenhui from the Huizhou Boluo County Agricultural Science and Technology Demonstration Farm advised us to pay attention to the long-term effectiveness of our products in protecting plants and whether there are any safety concerns regarding environmental residues.

Figure 9. Our visit to Huizhou Boluo County Agricultural Science and Technology Demonstration Farm.

Mr. Li Yonghong, a teacher at the Shenzhen Agricultural Science and Technology Promotion Center, advised us to pay attention to the safety issues associated with further experimentation on our products. He emphasized the importance of complying with current laws and regulations in both the laboratory and when expanding experiments. Ensuring compliance with legal and regulatory standards is crucial for conducting safe and responsible research and development activities.

Figure 10. Our visit to Shenzhen Agricultural Science and Technology Promotion Center.

To ensure that our products comply with the safety standards within the current legal framework, we conducted a detailed investigation into the requirements and regulations regarding pesticide use under the current laws. We compiled a document titled "Legal Analysis: Investigation into the Application of PolycoBead under Existing Laws" to summarize our findings. Through this investigation, we discovered that there are currently no specific standards or regulations in China regarding RNA pesticides. In order to better regulate this field and address the associated safety issues, we have drafted the "China RNA Pesticide Industry Standards Proposal" and submitted it to the relevant authorities.

To learn more, please click integrated human practices.