Safety
Becoming Responsible Scientists or Engineers

Guided by the spirit of iGEM, our team has innovatively developed a new type of sugar substitute production system aimed at benefiting a wider audience. At the project's outset, we recognized the importance of thoughtfully addressing potential risks to ourselves, our colleagues, the community, and the environment. Our commitment to safety and protection throughout the project has shaped us into responsible scientists and engineers.

We firmly believe that "working safely and reliably is a core element of responsible research and innovation". Therefore, we have focused our efforts on three key areas: Laboratory Safety, Project Safety, and Product Safety. We diligently adhere to laboratory guidelines and proactively identify potential risks in our experiments, implementing appropriate management measures. For our genetically modified crops, we have established a robust safety supervision system to ensure they pose no threat to humans or the environment. Furthermore, we comply with relevant laws and regulations and advocate for the importance of safety in practical human activities.

Lab Safety
Project Safety
Product Safety

General Lab Safety

Lab Configuration


Our experiments are conducted in the iGEM laboratory at Shenzhen University, which meets the standards of a Biosafety Level 2 laboratory and is equipped with facilities such as a fire protection system, access control system, electrical system, and safety alarm system, capable of meeting the needs of iGEM-related experiments.

The main functions and safety features of our laboratory include:

--Professional Plant Greenhouse: Our laboratory is equipped with a professional plant greenhouse that has good lighting and ventilation systems, meeting the needs for plant cultivation. As it is an enclosed space, it does not cause any harm to the environment.

--Laminar Flow Cabinet: It allows for experiments that require a locally clean and sterile working environment, such as sterile microbiological testing and plant tissue culture inoculation.

--Biosafety Cabinet: Our laboratory is equipped with a biosafety cabinet that meets national standards, which can effectively prevent the spread of hazardous or unknown biological particles during experimental operations, ensuring the safety of laboratory personnel and the environment.

--High-temperature sterilization and disinfection equipment and ultraviolet lamps: They can be used to disinfect equipment, instruments, reagents, etc., in the laboratory with ultraviolet light to prevent contamination and spread of hazardous substances.

--Emergency Equipment: Our laboratory is equipped with a range of emergency equipment, including but not limited to fire extinguishers, first aid kits, and eyewash stations, to enable self-rescue and protect our own safety in times of crisis.

--Waste Treatment Equipment: The laboratory is equipped with specialized organic waste liquid collection barrels, glass fragment recycling barrels, etc., to handle waste generated within the laboratory and prevent pollution and spread of hazardous substances.

--Protective Clothing and Gloves: Used to protect laboratory personnel from contamination and infection by hazardous substances.

Fig 1. Our laboratory and equipment.


Lab Safety Inspection


1. The laboratory conducts periodic safety inspections to identify and eliminate a range of laboratory hazard risk factors, and records these in the laboratory safety inspection log to ensure the safety of the laboratory. In addition, our Principal Investigator (PI) will supervise our experimental progress from time to time, and provide assessments and suggestions on the experimental procedures and results, ensuring safety throughout the experimental process.


Fig 2. Our Safety Checklist & Our Principal Investigator (PI) is discussing the progress of the experiment with us.

2. Various instruments and equipment in the laboratory are regularly inspected, cleaned, and maintained, and chemical reagents are periodically organized to ensure that chemicals, instruments, and experimental tools are stored according to prescribed methods, with labels indicating name, quantity, and expiration date, etc. For instruments and equipment that may pose a danger (such as those placed near electrical panels), we will rearrange their positions to ensure that safety issues do not arise due to electricity use.

3. At the same time, our Principal Investigator (PI) recommends that at least two people be present for each experiment. We will supervise each other during the experiment to ensure that all members conducting the experiment adhere to laboratory safety protocols, thereby reducing potential contamination risks. This is not only a responsibility for one's own safety but also for the safety of others and the environment, becoming a responsible scientist.

4. Our laboratory is equipped with emergency evacuation routes, first aid kits (medical first aid kits and AED, 30m from the laboratory), and shower facilities as emergency measures. During the experimental period, we will periodically check these fire and first aid measures to ensure that these facilities are valid and usable.

Fig 3. Our First Aid Equipment

5. During the experimental period, some of our infrequently used reagents and experimental supplies will be stored in a warehouse. We will also conduct safety checks on these items and regularly clean and organize them to ensure that they are neatly and orderly placed, eliminating all combustibles to prevent the occurrence of fires.



Personal Safety Protection


Laboratory Safety Skills Training


I. Laboratory Safety Knowledge Learning

Laboratory safety training is the cornerstone of laboratory safety, and everyone who enters the laboratory must be well-versed in it. In our freshman year, we are required to undergo laboratory safety training provided by the university. This includes studying and memorizing the laboratory safety manual, as well as taking corresponding tests. We must achieve a score of 90 or above in the exam to gain access to the laboratory.

Fig 4. Laboratory Safety Guidelines

In addition, we participate in lectures and practical activities related to laboratory safety to gain a more comprehensive understanding of laboratory rules and regulations. These measures ensure that we have a solid grasp of laboratory safety procedures and the necessary knowledge to conduct experiments safely.


II. Experimental Skills Training

Before conducting iGEM experiments, our team's Principal Investigator (PI) Liu Xuedong, and advisors Chen Ruoyu and Zhu Tangkun provided us with comprehensive training in basic laboratory skills. The experimental training covered a range of specific techniques, including PCR, agarose gel electrophoresis, Western Blot (WB), confocal microscopy, Agrobacterium infection, and more. They demonstrated the specific experimental processes and techniques to us and provided guidance and answered questions when we first attempted the experiments.

Fig 5. Our advisor is explaining experimental skills to us


In addition to specific experimental skills, the training also focuses on mastering laboratory preventive measures. These measures include, but are not limited to, strictly regulating the use of chemical reagents, carefully recording the use of equipment, and properly handling laboratory waste. PI Liu Xuedong specifically instructed that we must be familiar with the operating procedures and emergency measures before using dangerous equipment and reagents. Furthermore, he recommended a valuable laboratory book to us, which covers a range of topics, including but not limited to: how laboratories are organized, daily laboratory affairs, how to use a laboratory notebook and laboratory-related precautions. We have benefited greatly from it.





III. Assurance of Experimental Safety

During the experimental process, individuals who have not undergone laboratory training or who have not yet mastered the basic skills are not allowed to participate in experimental activities. In addition, each experiment must be conducted with a minimum of two people, who will supervise each other to ensure safety. Through these measures, we have increased the safety and reliability of our experiments, thereby reducing the risks that experiments may pose to ourselves, our colleagues, and the environment.

Fig 6. Our experimental scene.


Personal Safety Protection


In the laboratory, we must adhere to the corresponding codes of conduct to ensure our own safety.

Emergency Measures: Before conducting experiments, each team member must be familiar with basic emergency measures, including but not limited to how to use emergency facilities such as eyewash stations, how to handle fires, and knowledge of common safety signs. For hazardous chemicals involved in the experiment, it is necessary to know the appropriate response measures to avoid causing harm to oneself and others in the event of an accident.

Fig 7. Icons related to laboratory safety.

Personal Standards: Each team member must wear a lab coat, mask, and gloves, and tie back hair when conducting experiments. Eating and drinking are strictly prohibited in the laboratory, and wearing lab coats, gloves, or other protective equipment outside the lab area is not allowed.

Cleanliness: Upon entering and leaving the laboratory, everyone must clean themselves, such as washing hands and cleaning the experimental work surfaces.

Equipment Facilities: Each team member must be trained when using equipment. All devices must be strictly registered and used according to regulations. Before use, equipment should be inspected to ensure it is functioning properly; during use, one should always pay attention to the condition of the equipment to detect any abnormalities. During experiments, equipment should be cleaned and checked to prevent the leakage of residual hazardous substances.



Risk Estimation Management


Despite having received comprehensive laboratory training before conducting experiments, which has significantly increased everyone's safety awareness, accidents can still occur due to carelessness, potentially causing harm to oneself and others. Therefore, we have assessed potential risks in the laboratory and established corresponding measures to ensure our safety while working there.


Laboratory Organic Reagents


In order to prevent harm from organic reagents during experiments, we have conducted a safety check on all reagents used in the experiments and have implemented risk management for toxic and hazardous reagents (as described below) and established relevant emergency measures.

Substance Usage Harm Safeguard Procedures
Trizol Used for the extraction of total RNA in experiments. The main component of Trizol is phenol. Trizol helps to maintain the integrity of RNA during cell lysis and dissolution, making it very useful for the purification of RNA and the standardization of RNA production. Trizol reagent contains the toxic substance phenol, which can harm health due to inhalation, ingestion, or skin absorption. 1. Wear gloves during the experiment to prevent splashes.
2. If accidentally dropped on the skin, it should be immediately rinsed with a large amount of detergent and water.
Chloroform Chloroform, scientifically known as trichloromethane, is relatively stable and does not easily undergo chemical reactions. It is soluble in many organic compounds and can be effectively extracted from plant materials. It is primarily used for the extraction step in nucleic acid purification. It is irritating to the skin, eyes, mucous membranes, and respiratory tract. It is a carcinogen that can damage the liver and kidneys and is also a highly volatile gas. 1. Ensure wearing a mask, gloves, and safety goggles.
2. Perform operations inside a fume hood.
β-mercaptoethanol (BME) Used in the loading buffer for nucleic acid extraction or protein electrophoresis, the primary function is to break disulfide bonds. β-mercaptoethanol has an unpleasant odor. High concentrations of the solution can cause significant damage to the mucous membranes, upper respiratory tract, skin, and eyes. 1. Ensure wearing a mask, gloves, and safety goggles.
2. Perform operations inside a fume hood.
AS acetylsyringone Acetoin is a natural phenolic compound secreted by injured plants that can induce the activation of the Agrobacterium Vir genes, thereby promoting the integration of exogenous genes. By inducing the activation and expression of the Agrobacterium Vir region genes, it facilitates the processing and transfer of T-DNA, thus making it easier for the Agrobacterium T-DNA to enter the plant genome and integrate with it, improving the efficiency of plant transformation. AS (Acetosyringone) can irritate the eyes, respiratory system, and skin. 1. In case of eye contact, immediately flush with plenty of water.
2. If the situation is severe, seek medical attention immediately.
UV-light (ultraviolet light) Ultraviolet light is a common light source in laboratories, used for sterilization in laminar flow hoods. Ultraviolet light can damage the retina of the eye, and ultraviolet radiation is also a mutagen and carcinogen. Observation can only be made through filters or safety glass that absorbs harmful wavelengths. 1. Never look directly at an unshielded ultraviolet light source with the naked eye.
2. Use appropriate protective devices when turning on the ultraviolet light source.
3. Wear suitable protective gloves when operating under ultraviolet light.

Laboratory Operation

When pricked by a needle, the wound should be immediately cleaned, disinfected, and bandaged to prevent infection (the first aid kit is 30 meters away from the laboratory), and in severe cases, one should be sent to the hospital for timely medical treatment.
When frostbite occurs and is not severe, the affected area should be kept clean and dry immediately, and frostbite ointment should be applied. In severe cases, medical treatment should be sought promptly.
We should wear proper protective equipment such as safety goggles, masks, and gloves. Also, all operations should be conducted on ice to prevent shattering due to excessive temperature differences.
When burned, the wound should be cleaned and bandaged immediately. In serious cases, seek immediate medical attention; and when a fire breaks out, use appropriate firefighting measures to extinguish the fire immediately and call the police for help.

louis hide

Click to Project Safety
[1]Joseph JA, Akkermans S, Nimmegeers P, Van Impe JFM. Bioproduction of the Recombinant Sweet Protein Thaumatin: Current State of the Art and Perspectives. Front Microbiol. 2019 Apr 8;10:695. doi: 10.3389/fmicb.2019.00695. PMID: 31024485; PMCID: PMC6463758.
[2]Yoo SY, Bomblies K, Yoo SK, Yang JW, Choi MS, Lee JS, Weigel D, Ahn JH. The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta. 2005 Jun;221(4):523-30. doi: 10.1007/s00425-004-1466-4. Epub 2005 Jan 29. PMID: 15682278.
[3]Kurokawa, N., Hirai, T., Takayama, M. et al. An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin. Plant Cell Rep 32, 529–536 (2013). https://doi.org/10.1007/s00299-013-1384-7.
[4]Firsov A, Shaloiko L, Kozlov O, Vinokurov L, Vainstein A, Dolgov S. Purification and characterization of recombinant supersweet protein Thaumatin II from tomato fruit. Protein Expr Purif. 2016 Jul;123:1-5. doi: 10.1016/j.pep.2016.03.002. Epub 2016 Mar 8. PMID: 26965414.
[5]Masuda T, Okubo K, Baba S, Suzuki M, Tani F, Yamasaki M, Mikami B. Structure of Thaumatin under acidic conditions: Structural insight into the conformations in lysine residues responsible for maintaining the sweetness after heat-treatment. Food Chem. 2022 Sep 30;389:132996. doi: 10.1016/j.foodchem.2022.132996. Epub 2022 Apr 18. PMID: 35500407.
[6]Ruiz-Ojeda FJ, Plaza-Díaz J, Sáez-Lara MJ, Gil A. Effects of Sweeteners on the Gut Microbiota: A Review of Experimental Studies and Clinical Trials. Adv Nutr. 2019 Jan.
[7]EFSA Panel on Food Additives and Flavourings (FAF); Younes M, Aquilina G, Castle L, Engel KH, Fowler P, Frutos Fernandez MJ, Fürst P, Gürtler R, Gundert-Remy U, Husøy T, Manco M, Mennes W, Passamonti S, Moldeus P, Shah R, Waalkens-Berendsen I, Wölfle D, Wright M, Batke M, Boon P, Bruzell E, Chipman J, Crebelli R, Fitzgerald R, Fortes C, Halldorsson T, LeBlanc JC, Lindtner O, Mortensen A, Ntzani E, Wallace H, Civitella C, Horvath Z, Lodi F, Tard A, Vianello G. Re-evaluation of Thaumatin (E 957) as food additive. EFSA J. 2021 Nov 30;19(11):e06884. doi: 10.2903/j.efsa.2021.6884. PMID: 34876926; PMCID: PMC8630604.

During the brainstorming phase of our project, we aimed to do something interesting—creating a new production system for a novel Thaumatin [1] sugar substitute. The project is divided into two modules: the Expression System and the Storage System. We realized that our project could bring significant benefits to the world, but if not handled properly, it could also pose some potential risks and negative impacts. Therefore, we kept in mind iGEM's advocacy of "becoming responsible scientists or engineers" strictly adhered to iGEM safety requirements, and engaged in in-depth discussions with relevant stakeholders. We anticipated risks and formulated corresponding strategies, sincerely hoping that our project could bring greater benefits to humanity.


Organisms


During the initial phase of our experiments, we selected Escherichia coli BL21(DE3) for trials. Subsequently, we chose the common model organism Solanum lycopersicum (Micro-Tom) as our chassis and amplified the Tobacco rattle virus (TRV) [2] using Agrobacterium GV3101. We then injected the TRV virus expression vector carrying the Thaumatin gene fragment into tomato plants, successfully expressing the sweet protein in tomatoes. Additionally, we used Nicotiana benthamiana to verify the possibility of increasing the content of Thaumatin by adding the SPS-NTPP vacuolar sorting signal peptide to its N-terminus. To ensure the safety of our project and to guarantee that our experiments pose no harm to ourselves, our colleagues, or the environment, we conducted comprehensive research on the safety of the organisms used in the experiments.

Fig 1. Preventing Escherichia coli escape mechanisms.

As a commonly used model organism in scientific research, the safety of Solanum lycopersicum (Micro-Tom) and Nicotiana benthamiana is beyond doubt. Solanum lycopersicum (Micro-Tom) has been widely used by various bio-enterprises and researchers to explore gene function and regulatory mechanisms due to its relatively small and simple genome. Similarly, Nicotiana benthamiana , with its distinct genetic background, is also extensively used in plant research.

In addition, in order to ensure the safety of the strains that appear in the experiment, we have made the following attempts. First of all, we checked them in the iGEM whitelist, filled out the check in form as required, estimated the risk, and took corresponding measures to manage and reduce the risk. Secondly, we verified that they are prohibited from being used as infectious microorganisms in the Catalogue for the Control of Human Pathogenic Microorganisms. Escherichia coli BL21(DE3), Agrobacterium GV3101, and Tobacco rattle virus (TRV) viruses used in the laboratory are not on the list. Finally, the strains mentioned above are all commonly used strains in laboratory scientific research, and their safety can be guaranteed.

Escherichia coli BL21(DE3) is a non-virulent protein-inducible expression strain, derived from B-lineage Escherichia coli, which is widely used as a protein expression strain in experimental environments. Agrobacterium GV3101 is derived from Aspergillus tumefaciens biotype C58, which is commonly used to transform a variety of plants and allows for high transient expression; Tobacco rattle virus (TRV) virus is an engineered viral genome vector that can deliver partial sequences of target genes into plant cells. TRV is widely used for its high silencing effect, long duration of silencing, wide range of infiltrating host species, and mild symptoms of virus-induced disease.

Learn more in Part Collection.

Fig 2. Our strain is not on the list.

Our experiments are carried out in completely closed laboratories and are experienced in the use of these organisms. In addition, when dealing with laboratory waste, we strictly adhere to the relevant rules and regulations to ensure that these organisms do not leave the laboratory due to accidents or unforeseen factors.



Expression System


Sweet Protein


When it comes to food, food safety is always at the top of the list. This also shows that the safety of sweet proteins is crucial. Sweet protein is a sweet compound found in nature, mainly from plants in tropical Africa and Asia. These naturally derived proteins are so sweet, up to 100-1000 times more than sucrose, and do not cause significant health problems as well as calories compared to artificial sweeteners, there has been a lot of attention. Currently, eight sweet proteins have been identified, namely Thaumatin, Monellin, Mabinlin, Lysosyme, Pentadin, Brazzein, Curculin and Miraculin. Among them, Thaumatin stands out for its excellent strong stability, and is a more widely studied and used sweet protein.

Fig 3. Thaumatin and its source crop.

In communication with government regulators, we realized the need to comply with food-related laws and regulations and establish a sound safety system for Thaumatin to ensure its safety from origin to production and consumption, so as not to cause any health hazards to consumers, so that our new Thaumatin production system can be more easily introduced to the world.


Food Safety Evaluation

After communicating with Li Yonghong, the principal investigator from the Shenzhen Agricultural Promotion Center, we learned that genetically modified foods must undergo food safety evaluation before they are put on the market. According to the guidelines for the safety evaluation of genetically modified organisms formulated by the Codex Alimentarius Commission, the World Health Organization, the Food and Agriculture Organization of the World Food and Agriculture Organization and the Economic Cooperation Organization, food safety evaluation mainly includes nutritional evaluation, toxicological evaluation and allergenicity evaluation. Due to time constraints, we were unable to complete the food safety evaluation of Thaumatin in the laboratory in person, but we reviewed a large number of literature to ensure that Thaumatin was not pathogenic and allergenic.

According to the literature, Thaumatin has no adverse effects in rats and dogs for 90 days, and has no harmful effects in acute and subacute toxicity tests, teratogenicity, sensitization, mutation and immunity and other toxicity tests, proving its safety. In addition, Thaumatin does not cause tooth decay. And like other normal proteins, they are metabolized into corresponding amino acids in the intestine and then incorporated into normal intermediate metabolism, which has no significant effect on carbohydrate transport and metabolism, energy production and conversion, and amino acid transport.


Safety Certification

In 1979, Thaumatin was first approved for the market as a natural food additive in Japan. Since 1981, European countries have successively approved its use as a sweetener and flavor enhancer. Subsequently, the United States Food and Drug Administration (FDA) and the United States Food Flavors and Extracts Manufacturing Association (FEMA) deemed Thaumatin safe and approved for use in food[4]. In 2014, China officially approved Thaumatin as a food additive-sweetener (reference standard: GB2760-2014 National Food Safety Standard for the Use of Food Additives), which can be applied to frozen drinks, processed nuts, baked goods and beverages and other products. These certifications further indicate that Thaumatin is safe and reliable and not harmful to human health. At the same time, Thaumatin has been widely used all over the world, which has laid an important foundation for us to promote the new sweetener into the world market in the future, making entrepreneurship more feasible.

At the same time, when investigating sweet proteins, we focused on Brazzein, a small molecule sweet protein. Both in terms of stability and the sweetening effect it produces, Brazzein is not inferior to Thaumatin. However, although Brazzein has been consumed by indigenous Africans for centuries, its safety certification is still in the approval stage, which means that its safety cannot be guaranteed by strong laws. However, we all agreed that Brazzein would be a big surprise in the future. Therefore, we have also validated the expression of Brazzein in wet experiments (Learn more in Wet lab.) and hope that Brazzein will be approved for safety in the future, creating a richer world of sweetness.

Fig 4. Brazzein and its source crop.

Nature of Origin

Professor Mo Beixin from the Collage of Life Sciences and Oceanography at Shenzhen University told us that if we want to ensure the safety of Thaumatin, it is important to understand its origin. According to the data, Thaumatin is derived from the African arrowroot family, which is a class of proteins with a strong sweet taste isolated from its aril, and has no homology with other sweet proteins. Thaumatin's clean botanical background source means that it does not pose too much of a burden on human health, further guaranteeing the safety of Thaumatin. In addition, the plant origin of Thaumatin also lays the cornerstone for heterologous expression in tomato, which can achieve correct folding in tomato and reduce the safety risks caused by heterologous expression.

At the same time, Thaumatin consists of three domains (domains I, II, and III). Domain I (residues 1-53, 85-127, and 178-207) form the core domain and consists of 11 β chains[5]. Domain II is considered to be a large region rich in disulfide bonds, consisting of residues 128-177 and including four disulfide bonds and a helix. Domain III is considered to be a small region rich in disulfide bonds, consisting of residues 54-84, including two disulfide bonds and two key residues (Lys67 and Arg82), that elicit sweet taste sensations. At the same time, due to its 8 disulfide bonds, Thaumatin has strong thermal stability and strong acid resistance. This means that it is more conducive to subsequent food processing without cracking during processing and causing safety concerns.

Fig 5. Thaumatin structure diagram.

Express


Initial Expression Verification of Escherichia coli

Earlier in the experiment, we experimented with Escherichia coli BL21(DE3) to transfer the gene to the genome of the sweet protein Thaumatin and evaluate its risk. Our experiments will be carried out in a completely enclosed laboratory, and if it is not deliberately leaked or accidentally released, it will not cause any harm to the environment. In addition, we deliberately chose Escherichia coli BL21(DE3), an Escherichia coli that lacks Lon and ompT proteases. Once it leaves our medium, it dies, reducing the release of the organism. This means that even the initial attempt is safe.


Tomato Expression Verification

Fig 6. Tomato expression factor.

In our project, Agrobacterium infection was used to express Thaumatin in tomatoes and store them, aiming to create a novel sugar substitute production system. In tomato expression, we considered the following questions:

--Thaumatin is a heterologous protein for tomatoes, so may it affect tomato metabolism?


On the one hand, Thaumatin belongs to 22 kDa, a small molecule protein derived from plants. Because of its plant-derived background, its expression in tomato does not pose much metabolic burden and risk. And in recent years of scientific research experiments, researchers have successfully expressed Thaumatin in tomatoes, and its scientific research history can be guaranteed. On the other hand, in the experiment, we will continue to pay attention to the growth of tomato plants to see if their phenotype changes. Based on the time relationship, we only made superficial observations, but it is worth noting that the growth of the transgenic plants was almost identical to that of the control tomato plants.


--When using a novel promoter to induce Thaumatin expression, will the promoter affect the expression of Thaumatin in tomato?


When selecting promoters for inducible expression, we need to consider whether the promoter is safe, and whether the promoter can be stably expressed in tomato and passed on to offspring. Initially, we aimed at the CaMV 35S promoter. It is a 35S promoter derived from cauliflower mosaic virus (CaMV). As a constitutive promoter, it is able to initiate gene expression in all tissues with persistence. Therefore, in agricultural production, more than 80% of quasi-genetic crops approved for cultivation contain this promoter and have been proven to be safe, which also shows that the use of the 35S promoter in our early experiments is completely reliable. However, after communicating with Professor Mo, we noticed that the 35S promoter is a viral source after all, which cannot be safely inherited in offspring, and may affect the expression of the gene of interest in the transgenic gene. In addition, we need to store Thaumatin in vacuoles at vacuolar ripening (i.e., fruit ripening stage), while the 35S promoter does not exhibit spatiotemporal specificity, making it difficult to achieve precise localization.

Therefore, after talking with Prof. Huang Tengbo, a tomato expert from Shenzhen University, he asked us to aim at the strong promoter of the tomato itself. Therefore, we looked up the E8 promoter[3]. The E8 promoter is a specific ripening promoter derived from tomato and is often used to express foreign genes in tomato, and its safety is beyond doubt. Secondly, because of its maturation specificity, the E8 promoter can be well positioned to achieve vacuolar precision and reduce additional risks.


--Is Thaumatin expressed in tomato environmentally friendly?


From the promoter to our target sweet protein Thaumatin is of plant origin. Therefore, to a certain extent, the heterologous expression of Thaumatin in tomato is environmentally friendly and controllable. And the experiment is completely carried out in a closed laboratory, and there is no problem of accidental leakage.




Storage System


After talking with Li Yonghong, the principal investigator of the Shenzhen Agricultural Promotion Center, we learned that genetically modified foods need to ensure their stability before they can be approved for field trials. Therefore, in order to increase the content of Thaumatin in tomato and improve its stability in tomato, we added the vacuolar signal peptide - sweet potato sporozialmine and N-terminal propeptide (SPS-NTPP) to the N-terminus of Thaumatin, and stored Thaumatin in vacuoles through the vacuolar sorting decision cluster, which required us to discuss the safety of SPS-NTPP signal peptide, including but not limited to whether it can be safely expressed in tomato, whether it is safe to eat, etc. At the same time, we evaluated the safety of Thaumatin stored in vacuoles, fulfilling the promise of " being a responsible scientist or engineer ".


Fig 7. Thaumatin-Storage Roadmap .

Vacuole


Different strategies have emerged to improve the expression, yield, and structural stability of recombinant proteins, such as subcellular localization of recombinant exogenous proteins in various compartments of plant cells. Among them, vacuoles are one of the most common subcellular localization compartments. As the largest organelle in most plant cells, it plays various important roles in maintaining cell organization and function. Even vacuoles in some cells specifically accumulate storage proteins. We have ensured the safety of Thaumatin stored in vacuoles from the following two aspects:


From the perspective of sweet protein: Thaumatin is a protein with high thermal stability and high acid resistance, which can be stable for 30 mins in buffer with pH 2.0 and maintain sweetness. This illustrates the fact that Thaumatin can be safely stored in tomato vacuoles without being broken down into other substances. And even if it is broken down into amino acids in vacuoles, it will not cause much harm to human health.

From the perspective of vacuoles: As the largest organelle in tomato cells, vacuoles play various important roles in maintaining cell organization and function, and can even safely store exogenous recombinant proteins without negative effects. Therefore, storing Thaumatin in vacuoles is safe and stable to a certain extent. Tomato and tobacco are homologous plants, and the same is true in tobacco vacuoles.


SPS-NTPP


I. Source

Spore protein is a storage protein found in sweet potato (Ipomoea batatas) tuber roots and accumulates as a non-glycosylated monomeric protein in vacuoles of parenchyma cells. In addition, spore proteins are synthesized from membrane-bound multimers as precursors[6]. The N-terminal part of the precursor is the vacuolar signal peptide that is necessary to properly target it to the vacuole.

It can be seen that SPS-NTPP is also a plant source. A clean source of sweet potato plant background means that it does not cause much harm to human health and the environment. And as a protein containing 21 amino acid signal peptides and 16 amino acid propeptides (the 16 propeptides will be cleaved after translation), SPS-NTPP can be digested and broken down into the corresponding amino acids like other dietary proteins. However, we operate in a closed laboratory to ensure that we do not accidentally leak into the outdoor environment during the experimental phase.


II. Have A Scientific Research Background

After understanding the origin characteristics of SPS-NTPP, we are eager to know the safety of its heterologous expression to ensure that we can safely complete the validation experiments in tobacco. When this signal peptide is expressed in transformed tobacco calli, it has been reported that the precursor is correctly targeted to the vacuole without any negative effects. In addition, SPS-NTPP was successfully demonstrated to correctly express active recombinant human α(1)-protease inhibitor in tomato leaves, and did not change any morphology of tomato, and all transgenic plants were phenotypically normal, healthy and fertile with no adverse effects. This indicates that the vacuolar signal peptide SPS-NTPP we selected can safely complete the validation expression experiment in tobacco.



Exploration of Security Design



Based on our project is genetically modified tomatoes. Therefore, it is important to consider the ecological risks that may be brought to the environment when it is planted in the future. After reviewing the data, we learned that genetically modified crops may cause genetic drift when they are planted and produced, which may endanger ecological health. Gene drift refers to the transfer of genetic material (one or more genes) from one biological group to another biological population through a medium, which may lead to the decline of the purity of non-GMO seeds and the extinction of wild relatives. Therefore, it is very important to comprehensively understand the pathways of gene drift in quasi-genetic crops, estimate the risks, and formulate relevant measures to protect the ecological environment.

It has been reported that the pathways by which gene drift occurs are generally divided into 3 forms, depending on the medium:

Pollen mediation: the penetration of exogenous genes into other individuals through pollen is accomplished through natural hybridization, which mainly relies on the assistance of natural vectors of pollen transmission (such as wind and pollinators).

Seed-mediated: genetic drift that occurs through seed dispersal or dispersal, mainly with the help of natural media (such as wind, currents, animals) or human activities such as harvesting and transportation.

Probesan-mediated: It mostly occurs in perennial plants, and is a genetic drift transmitted by plant propagules, mainly with the help of natural media (such as water flow or animals) or human activities.


Considering the practical project and theoretical knowledge, we explored the possible genetic drift of genetically modified tomatoes in the project. First of all, during planting, our seeds undergo strict management and are difficult to expose to the natural environment. At the same time, plants grown in greenhouses do not meet the conditions for propagules-mediated exposure. Therefore, it is unlikely that gene exposure will be achieved through seed-mediated as well as propagular-mediated pathways.

However, pollen gene drift is the main pathway for gene drift. And whether it is seed-mediated or propagule-mediated, it is ultimately through pollen hybridization that the gene exchange between individuals can be truly realized. Therefore, we have shifted our focus to pollen-mediated genetic drift.

Because pollen-mediated genetic drift is accomplished by both crops and the environment. Therefore, in order to provide a more rational and comprehensive solution, we explored a solution to prevent genetic drift from the environment itself and the crop itself, and rationally verified its feasibility.


--From the Environment:

Pollen-mediated genetic drift relies heavily on natural mediators, such as wind and pollinators. But then we found it difficult to develop a response from wind to pollinators. On the one hand, it is difficult for us to control abiotic natural factors such as wind; On the other hand, pollinators are very diverse and abundant in nature. Therefore, it is obviously very unwise to develop relevant management measures based on wind and pollinators.

However, we also note that it is not impossible to start with the environment. At present, there are also some physical measures to prevent pollen-mediated gene drift, taking into account the geographical location and natural environment of transgenic crop planting, and setting up certain spatial isolation, such as setting blank rows and setting up isolation nets between transgenic and non-transgenic crops. Based on the current physical prevention methods, we intend to set up anti-bird nets in the test site to isolate non-GMO crops to prevent genetic drift.


--From the Crop Itself:

At present, in order to prevent genetic drift caused by pollen, a number of control methods have emerged[7]:

  • Cytoplasmic transgenic technology: By introducing exogenous genes into the genetic system of the cytoplasm (usually mitochondria or chloroplasts), exogenous genes can only be inherited through the mother, thereby reducing or even blocking the pollen-mediated gene drift of exogenous genes.

    • Advantages: Pollen-mediated gene drift is cut off at root.

    Disadvantages: However, plastid DNA may be transferred to pollen, chloroplast DNA may be integrated into the nuclear genome, and it is technically difficult to achieve the desired effect


  • Male sterility technology: The introduction of exogenous genes into male sterile individuals or through other molecular means to make the crops with exogenous genes become male sterile individuals, resulting in the inability of transgenic crops to produce pollen or fertile pollen.

    • Advantages: This method does address the root cause of pollen-induced genetic drift.

    Disadvantages: However, this method causes pollen sterility, difficulty in fruit formation, and inability to produce the target product, Thaumatin protein.


  • Cleistogamy restriction technology: Pollen is directly transferred to the pistillation of the same flower or pollen is directly germinated in the pollen sac, and fertilization can be completed without flowering.

    • Advantages: It is indeed effective in preventing pollen-mediated gene drift in closed-pollinated crops.

    Disadvantages: It is difficult to achieve in all crops, especially for alienated pollinated crops.


  • Gene excision technology: In the process of male gamete formation, transgenic fragments are excised from the genome by recombinant enzymes to form male gametes without transgenic components.

    • Advantages: In the process of male gamete formation, transgenic fragments are excised from the genome by recombinase to form male gametes without transgenic components.

    Disadvantages: There will still be a recognition site of recombinase after gene excision, and there will be transgenic components, and it is not 100% confirmed that complete excision is complete, and its safety is difficult to guarantee.





    Our Design

    At the beginning, we planned to make trait differentiation so that we could distinguish genetically modified tomatoes when planting, and at the same time, we could report to the relevant departments for management after genetic drift of genetically modified tomatoes was discovered. However, in the process of human practice, we have found that the differentiation of phenotypes cannot fundamentally solve the problem of leakage caused by pollen transmission. It's not a very good idea. Therefore, we abandoned this idea.

    Then we looked at the projects that the iGEM community has been working on genetically modified crops in previous years, and among them, we were inspired by the Stony_Brook (United States) team, who used optogenetic kill switches to prevent genetic drift. Therefore, we borrowed their design as our prevention switch, using the tetracycline UVR8-COP1 system to initiate transcription of downstream RNAi. UVR8 is a plant photoreceptor responsible for regulating UV-B-triggered signaling pathways. COP1 is a key regulator of photomorphogenesis. UVR8 senses UV-B through tryptophan residues (Trp 233/285). Under UV-B irradiation, UVR8 underwent a conformational change and completely dissociated, exposing 27 residues C-terminal extension (C27) that binds to COP1 interdependently. UVR8 is fused to TetR (also known as the tetracycline repressor domain), which maintains binding to the tetO consensus sequence, and the VP16 activation domain is fused to COP1. When the UVR8-COP1 pair is not bound, transcription does not occur. When irradiated with UV-B, COP1-VP16 is recruited to the nucleus, initiating transcription of downstream genes. We designed a silencing RNAi sequence for WUSCHEL (WUS) downstream, and the WUS gene plays a key role in regulating the coordination of cell proliferation and differentiation. Once the transgenic crop is exposed to the wild, it activates gene silencing of WUS, thereby inhibiting the growth and development of the plant.

    Based on the above discussion, we propose a means to prevent genetic drift for our own transgenic tomatoes. We achieve this goal in two ways: on the one hand, we can solve the problem of gene leakage by restricting the environment of our genetically modified crops and planting them in pilot sites that have been approved for genetically modified planting. On the other hand, we intend to introduce the tetracycline UVR8-COP1 system into transgenic tomatoes to initiate downstream RNAi inhibition of the WUS gene. When genetically modified tomato pollen leaves our specially set environment, it is exposed to UV-B to induce RNAi and prevent gene drift.

    Fig 8. Pollen Suicide Switch Schematic Diagram.

    Discussion

    We then submitted the idea to the school's GMO safety team and the local agricultural department, only to find that this method was of little use and that we could achieve the effect of physical control. It includes the following sections:

    --First of all, our genetically modified tomatoes are available to the general public as food. Therefore, adding such a complex and bulky suicide switch system to the tomato genome poses a greater GMO problem.

    --Secondly, from the level of practical conditions. At present, China has relatively good physical control measures for genetic drift caused by genetically modified crops, such as intercropping and planting on land containing anti-bird nets, which have achieved good results. In addition, Shenzhen University has a test base approved by the state that meets the national standards, and the test base is equipped with anti-bird nets and other equipments, which can fully meet the needs of isolation.

    --It is difficult to achieve precise regulation in the setting of specific environments, which undoubtedly increases the difficulty and cost of genetically modified crop planting.

    --Finally, our target gene is derived from plants, which is a protein that is harmless to the environment and crops, so from this point of view, it will not cause harm to the environment.


    After listening to the opinions of all parties, we took a closer look at our design. We believe that designing pollen safely will only make our GM tomatoes more unsafe. Therefore, we intend to keep the physical measures and abandon the design for tomato pollen. But we hope that our idea can provide some inspiration for the team that intends to make genetically modified crops in the future, combine physical and biological measures, and design more reasonable and safer designs for genetically modified crops.



    Risk Estimation Management


    Project Risk Assessment


    Although the Thaumatin, E8 and SPS-NTPP we used in the project species are all derived from plants, and the strains used are also commonly used in scientific research experiments, and their safety can be guaranteed, we still evaluated the project risks and found that:

    --- Our experiments are carried out in the secondary closed laboratory, and the experimenters are strictly dressed according to the requirements when entering and leaving the laboratory. As a result, there is hardly any question of exposure.

    -- Our laboratory has been approved by the GMO Committee for safety, has a special plant room to grow our GM tomatoes, and the tomato waste will be strictly treated. Therefore, there will be no problem of pollen drift in genetically modified crops.

    -- Escherichia coli, Agrobacterium, and viral vectors are difficult to survive after leaving the laboratory, and genetic contamination almost does not occur. And the harm to the human body is very small. As a result, even when exposed to the environment, the risk to public health, agriculture and the environment remains small.

    To sum up, the benefits of our project outweigh the disadvantages. And we carried out a risk assessment to further ensure the safety of the project.


    Information and Technology Risk Assessment


    In this project, there is little risk that knowledge, information and technology may be misused. All the methods used in this project are basic molecular biology techniques and basic cell culture techniques, including agarose gel electrophoresis, WB, Agrobacterium infection, etc. The digital tools used are from the public domain and are widely used in agricultural GMO production. The project will not develop any new technology that may pose a risk of abuse, nor will it use the knowledge, information, technology, or products generated by research to harm people, crops, the environment, the economy or safety. Nor does the misuse of these technologies have serious consequences for public health, agriculture, the environment, the economy, or terrorism.

    louis hide

    Click to Product Safety
    [1]Joseph JA, Akkermans S, Nimmegeers P, Van Impe JFM. Bioproduction of the Recombinant Sweet Protein Thaumatin: Current State of the Art and Perspectives. Front Microbiol. 2019 Apr 8;10:695. doi: 10.3389/fmicb.2019.00695. PMID: 31024485; PMCID: PMC6463758.
    [2]Yoo SY, Bomblies K, Yoo SK, Yang JW, Choi MS, Lee JS, Weigel D, Ahn JH. The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta. 2005 Jun;221(4):523-30. doi: 10.1007/s00425-004-1466-4. Epub 2005 Jan 29. PMID: 15682278.
    [3]Kurokawa, N., Hirai, T., Takayama, M. et al. An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin. Plant Cell Rep 32, 529–536 (2013). https://doi.org/10.1007/s00299-013-1384-7
    [4]Firsov A, Shaloiko L, Kozlov O, Vinokurov L, Vainstein A, Dolgov S. Purification and characterization of recombinant supersweet protein thaumatin II from tomato fruit. Protein Expr Purif. 2016 Jul;123:1-5. doi: 10.1016/j.pep.2016.03.002. Epub 2016 Mar 8. PMID: 26965414.
    [5]Masuda T, Okubo K, Baba S, Suzuki M, Tani F, Yamasaki M, Mikami B. Structure of thaumatin under acidic conditions: Structural insight into the conformations in lysine residues responsible for maintaining the sweetness after heat-treatment. Food Chem. 2022 Sep 30;389:132996. doi: 10.1016/j.foodchem.2022.132996. Epub 2022 Apr 18. PMID: 35500407.
    [6]Ruiz-Ojeda FJ, Plaza-Díaz J, Sáez-Lara MJ, Gil A. Effects of Sweeteners on the Gut Microbiota: A Review of Experimental Studies and Clinical Trials. Adv Nutr. 2019 Jan.
    [7]EFSA Panel on Food Additives and Flavourings (FAF); Younes M, Aquilina G, Castle L, Engel KH, Fowler P, Frutos Fernandez MJ, Fürst P, Gürtler R, Gundert-Remy U, Husøy T, Manco M, Mennes W, Passamonti S, Moldeus P, Shah R, Waalkens-Berendsen I, Wölfle D, Wright M, Batke M, Boon P, Bruzell E, Chipman J, Crebelli R, Fitzgerald R, Fortes C, Halldorsson T, LeBlanc JC, Lindtner O, Mortensen A, Ntzani E, Wallace H, Civitella C, Horvath Z, Lodi F, Tard A, Vianello G. Re-evaluation of thaumatin (E 957) as food additive. EFSA J. 2021 Nov 30;19(11):e06884. doi: 10.2903/j.efsa.2021.6884. PMID: 34876926; PMCID: PMC8630604.

    Considering that our project is to use genetically modified tomatoes to produce sweet proteins, we have made efforts to ensure the safety of genetically modified tomato cultivation and genetically modified foods in the following aspects. In a wide range of human practice activities, we have collected laws and regulations related to genetically modified crops, exchanged and discussed with plant experts, conducted field investigations, and formulated relevant laws and regulations and risk management manuals, so that our new sugar substitute production system can enter people's vision in a safer way.


    Genetically Modified Crops


    Present Regulation


    Interpretation of Legal Instruments & Guidelines

    In China, the management of genetically modified crops involves several laws and regulations. Together, these laws and regulations form the legal framework for the management of genetically modified crops in China, aiming to ensure the safety and legality of genetically modified crops, and to protect consumer rights, biodiversity and environmental safety. The following laws and regulations have become our guiding principles for the subsequent promotion of GMOs into the market, and by interpreting these policies, we can better understand which directions we need to work towards:

    Name Regulation Response
    Regulations on the Safety Administration of Agricultural Genetically Modified Organisms China's core regulations on GMO safety management cover the safety management of the research, testing, production, processing, management, import and export of GMO crops. After reading this regulation, we have a preliminary understanding of the safety of genetically modified crops. We then communicate with experts and conduct site visits to further estimate and manage risks.
    Administrative Measures for Labeling of Agricultural Genetically Modified Organisms The measures stipulate the labeling requirements of genetically modified agricultural products and their processed products to protect consumers' right to know and choose, and ensure that genetically modified products on the market can be clearly identified. In order to ensure consumers' right to know and choose, and to distinguish genetically modified crops from normal crops, it is necessary to add labels to genetically modified foods. Subsequently, we visited the company in the field and expected to indicate their GMO source in our GMO products.
    Environmental Protection Act Although it is not a law specifically aimed at GMOs, it stipulates the basic requirements of environmental protection, and the environmental release and cultivation of genetically modified crops need to comply with the relevant provisions of environmental protection. During the experiment of transgenic in the experimental field, gene leakage caused by pollen may cause harm to other crops in the environment. Therefore, we conducted research in this aspect and tried to design a pathway to prevent gene drift, so as to ensure environmental safety.

    Talk to Experts


    Professor Mo Beixin

    After interpreting the existing laws and regulations related to GMO, in order to further understand the specific process and precautions of GM crops from the laboratory to the test base, we visited Professor Mo Beixin from the Collage of Life Sciences and Oceanography of Shenzhen University. Professor Mo told us that if the transgenic crops in the laboratory are to successfully and safely enter the test site, they must ensure that the target gene is stably expressed in the crop. In addition, we expressed our concerns to Professor Mo about genetic drift caused by the escape of genetically modified pollen, and asked if it was possible to control pollen through synthetic biology. Professor Mo told us that on the one hand, if pollen is designed, it will increase the metabolic burden of tomatoes, and the gains outweigh the losses. On the other hand, physical isolation, such as the installation of anti-bird nets, can effectively prevent pollen from causing genetic drift. Finally, Professor Mo recommended that we visit the base of the Bioindustry Innovation Research Institute of Shenzhen University to gain a better understanding of the current means to prevent GM drift.



    Associate Professor Liu Yun

    After the design of the project was completed, we had the opportunity to have a detailed communication with Associate Professor Liu Yun, a professor of botany. She raised a key safety concern, noting that the hygromycin resistance gene used in the initial design of our project may produce hygromycin protein during expression. Associate Professor Liu Yun emphasized that genetically modified crops cannot carry fragments other than effective genes, and if this protein is ingested by the human body, it may cause adverse effects on the health of consumers. In response to this important feedback, our team re-evaluated the safety of the hygromycin gene.




    Fieldwork Visit


    BGI

    After communicating with the professors, we visited BGI in the field. As the founder of China's gene industry, BGI has explored high-quality genes and cultivation applications that affect crops on a large scale. When they learned that our project was to produce the sweet protein Thaumatin using genetically modified tomatoes, they showed great interest. At the same time, they also emphasized the need to pay attention to the safety of exogenous genes, and always pay attention to the expression of sweet protein Thaumatin in tomato, such as whether it will affect the metabolism of tomato itself.



    Shenzhen Agricultural Promotion Center

    Concerned about the use of Agrobacterium infection, we visited the Shenzhen Agricultural Promotion Center. Principal investigator Li Yonghong from the Promotion Center suggested that we should not consider this issue because if genetically modified crops are commercialized, Agrobacterium infection will not be used. She suggested that we should focus on the expression and stability of Thaumatin in tomatoes, and provide consumers with recommended intake. At the same time, she also introduced to us the current management measures of genetically modified crops, on the one hand, the review of genetically modified crops from the two dimensions of food safety and environmental safety; On the other hand, there is the management of genetically modified organisms. Successful listing is only allowed after three steps: pre-approval, intermediate testing and production testing.

    In addition, Ms. Li emphasized the importance of complying with current laws and regulations in the laboratory. Ensuring compliance with legal and regulatory standards is essential for conducting safe and responsible R&D activities.



    Genetically Modified Experimental Base of Shenzhen University

    After studying the "Compilation of Supporting Laws and Regulations for the Safety Management of Agricultural Genetically Modified Organisms", we learned that after obtaining reliable results in laboratory research, agricultural genetically modified crops still need to go through three stages: intermediate experiments, environmental release and production tests. Therefore, we visited the base of the Bioindustry Innovation Research Institute of Shenzhen University to understand the growth of genetically modified crops in the experimental stage and learn the corresponding safety protection measures.

    In the course of the conversation with Mr. Lou, the person in charge of the base, we learned that in order to avoid the impact of the unexpected spread of genetically modified crops on the natural environment, the base has taken a series of special measures, such as planting the same species only 300 meters away from the genetically modified crops, and setting up anti-bird nets around the experimental field to prevent birds from eating seeds, and setting up 24-hour surveillance cameras to prevent outsiders from entering. In addition, he introduced us to the smart greenhouse, which prevents genetic drift caused by pollen through waterless cultivation and smart spraying system. These strict management measures are aimed at protecting the ecological environment and ensuring the reliability of scientific research, thus further guaranteeing the possibility of GM tomatoes coming out of the laboratory in the future.




    Risk Prediction and Management


    After interpreting legal documents, communicating with experts and professors, and conducting field visits, we have a deeper understanding of the safety of genetically modified crops. And we have thought deeply about our project, and have made efforts to ensure the safety of genetically modified tomatoes in the following aspects:

    Ⅰ. Project Safety

    In multi-party interviews, we summarized several issues that need attention in order to ensure the safety of GM crops and considered relevant strategies:

    -- Can the target gene be stably present and expressed in transgenic tomatoes?

    It has been reported that when the tomato matures, the internal pulp cells are fragmented, resulting in the outflow of intracellular organelles that fill the entire interior of the fruit. At this time, the slurry is filled with various enzymes that flow out as the cells are broken. If sweet proteins are stored in the cytoplasm, they will be difficult to stabilize. So, we aimed at the vacuole. As an organelle with a large maturation stage and few enzyme systems, it is a good place to store heterologous expressed proteins.

    Therefore, we designed the SPS-NTPP vacuolar signal peptide at the N-terminus of sweet protein to enhance the stable expression of sweet protein Thaumatin in tomato. When the tomato is ripe, the vacuoles gradually ripen and occupy most of the space of the cell. Through vacuolar targeting, we may be able to achieve the conditions for stable expression of the target gene in transgenic crops, so as to ensure the safety of subsequent transgenic crops into the experimental base.


    --Is there a standard-compliant testing site with physical isolation to prevent gene drift?

    During the conversation with Professor Mo, we learned that in order to strengthen the safety management of quasi-GMOs at Shenzhen University, Shenzhen University has set up a GMO safety team to supervise matters related to GMO work at Shenzhen University. In addition, Shenzhen University has its own experimental base and has obtained the approval of GMO from the Agricultural GMO Safety Management Office of the Ministry of Agriculture and Rural Affairs. It can be seen that Shenzhen University can provide a test base with physical isolation that meets the standards for transgenic tomatoes, and takes safety control measures suitable for safety registration level II to carry out relevant tests.

    --Is the hygromycin tag added to screen for GM crops harmless during the experimental phase?

    Subsequently, we conducted an in-depth investigation on the safety of the resistant tag hygromycin to ensure that hygromycin is safe and harmless even in the experimental stage, and will not cause harm to the human body, colleagues and the environment. Hygromycin B is an aminoglycoside antibiotic isolated from Streptomyces hygroscopicus, HPT is a hygromycin resistance gene derived from Escherichia coli.

    It has been reported that hygromycin resistance genes have strong specificity and are often used in scientific research to develop clones of higher organisms. At the same time, the HPT-expressed protein had no adverse effects in the study of acute oral toxicity in mice, and the results showed no adverse reactions to subchronic toxicity test, immunotoxicity assessment, teratogenicity study and nutritional evaluation of transgenic rice containing HPT. In summary, HPT protein, as a selective marker of transgenic rice, will not cause any harm to human health and the environment.

    Even if hygromycin labels are harmful, China's relevant laws and regulations stipulate that all resistance labels must be removed when transgenic crops enter the test base and go on the market, so the safety of transgenic tomatoes on the market is also guaranteed.


    Ⅱ. Manual Writing

    After reading the laws and regulations related to GMO, we found that the current GM laws and regulations are aimed at the relatively large GMO background, without specific operational guidelines, and the boundary between GM crops and synthetic biology crops is not clear. Therefore, in order to better adapt to our project and provide a more detailed guiding law for the new sugar substitute system, we have written The Biosecurity and Bioethics Whitepape and Genetically Modified Crop Assessment and Development Manual for the legal analysis of genetically modified foods


    The Biosecurity and Bioethics Whitepaper

    The Biosecurity and Bioethics Whitepaper provides a comprehensive introduction to the Food and Nutrition track in the iGEM competition, including an overview of the project, track background, analysis of bioethics and safety regulations, and discussion of biosecurity measures. In The Biosecurity and Bioethics whitepaper, SZU-China highlights the need to responsibly produce synthetic biology foods. In terms of bioethics and regulations, The Biosecurity and Bioethics Whitepaper explains in detail the differences between domestic and foreign food safety regulations and puts forward suggestions for the improvement of future regulations. In addition, the documentation highlights the unique design of the team's project, including innovation and feasibility.

    Overall, this white paper is a comprehensive report that aims to showcase the team's work in the field of Food and Nutrition and highlight the importance of following bioethics and safety regulations in biotechnology applications.


    Genetically Modified Crop Assessment and Development Manual

    On the basis of in-depth research and compliance with the current national laws and regulations on genetically modified crops, our team has carefully drafted a legal document on genetically modified crops, aiming to provide clear specifications and guidance for the cultivation of genetically modified crops. This legal document details the stages of GM crops from research and development, field trials to commercial cultivation, ensuring that cultivation activities are carried out within the framework of strict biosafety and ethical standards. The purpose of this law is to promote the responsible use of GM technology by clearly defining planting conditions, risk assessments, regulatory measures and public participation mechanisms, while safeguarding environmental safety, public health and biodiversity.



    Product -- Sweetein


    Present Regulation


    Interpretation of Legal Instruments & Guidelines

    In China, the management of food safety involves a number of laws and regulations. Together, these laws and regulations constitute the legal framework for food safety management in China, and guidelines and industry standards have been formulated. The following laws and regulations have become our guiding guidelines for the subsequent promotion of genetically modified foods into the market, and through the interpretation of these policies, we have a better understanding of the direction in which we need to go:

    Name Regulation Response
    Food Safety Law
    Regulations for the Implementation of the Food Safety Law    		 Although the Food Safety Law is not a specific law for genetically modified food, it stipulates the safety requirements for the production and sale of food, and genetically modified food, as a type of food, also needs to comply with the provisions of the law.
    The Regulations for the Implementation of the Food Safety Law further clarify some provisions in the Food Safety Law, including specific requirements for food production and operation, and put forward more detailed requirements for food labels and instructions to ensure that consumers can obtain sufficient food information.     		After having a certain understanding of food safety law, we knew that food safety is very important, and had a deeper reflection on the safety of sweet protein in our project.
    Measures for the Administration of Food Safety Sampling Inspection For genetically modified and synthetic biology foods, this approach requires rigorous sampling tests to assess their safety. We visited food-related companies such as Yuanqi Forest and Besheng Biotech, learned about their sampling methods, and formulated a regulatory manual dedicated to sweet protein to ensure the safety of products from production line to market.
    Convention on Biological Diversity The impact on biodiversity during the research and preparation of synthetic biological foods is governed by the Convention on Biological Diversity, which came into force in 1993. When carrying out research and application of synthetic biological foods, States Parties shall comply with the requirements of the Convention and undertake treaty obligations for the protection of natural biological resources and sustainable development. We strictly abide by the Convention on Biological Diversity in our experiments, and the whole process of the experiment is carried out in a closed laboratory to ensure that synthetic biological foods will not be leaked. In addition, we participated in the Biosafety Exhibition to promote synthetic biology safety.

    Talk to Expert


    Professor Zhao liqing

    At the same time, in order to have a deeper understanding of the safety requirements of food additives and the specific process from food to market, we had an online exchange with Zhao Liqing, a teacher majoring in food from Shenzhen University, to further consider food safety in our project. Mr. Zhao introduced to us the current industry standard for food additives (GB 2760-2022 Standard for the Use of Food Additives), which should not reduce the nutritional value of food itself if it does not cause any health hazards to the human body. In addition, she also introduced us to the standard requirements for food produced in laboratories and the current food safety regulatory system in China. We understand that if laboratory food wants to enter the market, it must go through toxicological evaluation, allergenicity evaluation, key ingredient analysis and nutritional evaluation, etc., which further inspired us to pay attention to the safety of Thaumatin.


    Due to experimental conditions and time constraints, we are unable to complete all food safety regulatory processes. However, we have conducted as many thorough investigations as possible on Thaumatin to ensure that it does not pose a risk to human health, and hope that the regulatory process can be completed gradually.



    Fieldwork Visit


    CHI Forest

    In order to fully understand the current processing and approval process of sugar substitute foods and the food safety evaluation criteria, we visited the Zhaoqing factory of Yuanqi Forest, a well-known sugar substitute beverage company in China. In order to ensure the safety of sugar substitute beverages, they have developed a sound regulatory system, including the selection of sugar substitutes, the establishment of internal laboratory safety standards, the establishment of a food safety laboratory to conduct random inspections of beverages at all stages, and the "one-vote veto" germination of responsible product hygiene.

    After the visit, we were inspired. Considering that even if Thaumatin is derived from plants and has obtained FDA edible certification, its long-term safety is difficult to guarantee, therefore, it is urgent to establish a one-stop supervision system for Thaumatin from production to marketing to after-sales, formulate exclusive food standards, assess risks and supervise from time to time, and strangle food hazards at birth.



    Eubiotic Organisms

    In order to gain an in-depth understanding of the application of synthetic biology technology in the food industry and the safety requirements for its market access, our team made a special trip to Besheng Biotechnology Co., Ltd. for consultation. In a professional consultation with Ms. Liu, the head of the company, we discussed in detail the safety standards and regulatory processes that synthetic biology technical foods must follow before they are marketed. This meeting made us realize that the commercialization process of synthetic biology technology foods involves rigorous regulatory review, including but not limited to key aspects such as product risk assessment, safety testing, labeling specifications, and post-market surveillance. Ms. Liu also introduced the company's advanced measures and innovative practices in ensuring the safety of synthetic biotechnology food. Through this fruitful exchange, we gained a more comprehensive understanding of the market access requirements for synthetic biology technical foods and provided valuable reference information for the further planning and implementation of our project.



    Food Event Exhibition

    In addition to that, we visited the SIAL International Food & Beverage Exhibition. As the world's leading event for the food and beverage industry, it brings together top brands, innovative technologies and cutting-edge ideas from around the world. Here, we talked to various food suppliers, learned about current industry standards for food safety, and marketed guidelines, and they emphasized the importance of focusing on the safety of the food itself and the processing process, which inspired us to pay more attention to the safety of the sweet protein Thaumatin itself and the safety of the processing process. At the same time, we saw that the GM food on the stall had the GMO logo, which inspired us to design our product packaging to distinguish it from other non-GMO foods (see discussion below).




    Risk Prediction and Management


    After interpreting legal documents, communicating with experts and professors, and conducting field visits, we have a deeper understanding of food safety, and have thought deeply about the sweet protein in our project, and have made efforts to ensure food safety in the following aspects:

    I. Project Improvement

    After the discussion, we realized that the safety of sweet proteins is critical to our project, and it is not enough to know its origin and FDA certification. Therefore, we have conducted a deeper safety assessment of sweet protein in accordance with the laws and regulations related to GM food safety to ensure the safety of sweet protein Thaumatin from different dimensions.




    II. Formulate Laws and Regulations
    Genetically Modified Food Long-term Safety Regulatory Manual

    Based on a comprehensive review and analysis of the existing regulations on the production and post-marketing supervision of genetically modified products in various countries, SZU-China has compiled a professional manual called "Food Long-term Safety Supervision Manual". The handbook details our team's regulatory framework for GM products from planting, testing, production to final post-market development, ensuring high standards of product safety and efficacy. The Genetically Modified Food Long-term Safety Regulatory Manual covers multiple key aspects such as risk assessment, quality control, labeling, market surveillance, and emergency response, and aims to provide clear guidance and reference for regulatory authorities, producers, and consumers. Through the implementation of this system, we are committed to building a transparent, efficient and dynamic regulatory environment to promote the healthy development of GM technology, protect the public interest, and promote agricultural science and technology innovation.


    Product User Manual

    Our team has created a Product User Manual designed specifically for consumers to provide a comprehensive overview of our genetically modified tomato sugar substitute products. In plain language, this handbook explains in detail the scientific basis of tomato sugar substitutes, their health benefits, and how to incorporate them into your daily diet. We emphasize the safety of our products, from genetic selection to the production process, every step of the process is strictly controlled and supervised to ensure that consumers can enjoy it with peace of mind.

    The handbook also contains a wealth of recipes and serving suggestions on how to use tomato sugar substitutes to enhance the flavor of food while controlling sugar intake. Through transparent disclosures and consumer education, we are committed to helping you make informed food choices and enjoy a healthy, delicious modern life. The Product User Manual is a reflection of our responsibility to consumers and our ongoing commitment to product transparency and consumer trust.


    III. Public safety

    The Implementation Regulations of the Food Safety Law stipulate that the public has the right to be informed and choose genetically modified foods. In order to ensure that our products can be distinguished from other non-genetically modified foods, we drew inspiration from the food exhibition. We have carefully designed the product packaging to indicate that it is a genetically modified product, ensuring that the public has the right to be informed. In addition, we have considered the needs of special individuals (such as blind people) and designed braille on the product packaging to facilitate blind identification.

    Our product and braille design


    Safety Promotion


    We know that safety depends not only on us, but on millions of people. Therefore, we not only aim at our project, but also hope to arouse the safety awareness of the society, popularize the safety and ethical issues brought by biotechnology, and implement safety in all aspects of life.


    Laboratory Safety Science Video


    In order to emphasize the importance of laboratory safety and let more students pay more attention to laboratory safety and abide by laboratory safety guidelines, we have filmed a laboratory safety science video and presented it in front of people in an entertaining and educational way. We hope that through this popular science video, more people can understand and learn about the safety rules of the laboratory, and promote the construction of a safe laboratory.

    Our laboratory safety science video



    Project Safety Promotion


    Biological Public Safety Exhibition

    At the same time, we take into account the people in the community. For them, the biggest concern is undoubtedly the safety and ethical issues posed by new technologies. Therefore, in order to better dispel the fears and worries of the public about synthetic biology and educate the public about the biosafety of the iGEM project, we participated in the biosafety exhibition held by Southern University of Science and Technology in Nantou Ancient Town, Shenzhen, aiming to promote safety to everyone's heart. At the exhibition, we answered questions about biotechnology safety and focused on the safety of genetically modified crops in our project, which strengthened the public's trust and understanding of biotechnology projects.



    Sugar Substitute Science Picture Book

    In addition, we pay special attention to the youth group. Due to the lack of systematic safety education, their awareness of food safety is relatively shallow. Therefore, we have drawn a popular science picture book on sugar substitutes, aiming to enhance the attention of young people to food health. The picture book shows the current classification and safety of sugar substitutes in an entertaining and educational way, and emphasizes food safety in daily life. In this way, we hope to bring food safety into the daily lives of young people in a more interesting way.

    Our sugar substitute science picture book


    Safety in Human Practices


    (1) In the human practice carried out by SZU-China, we collected questionnaires about sugar substitutes from the masses. Once the questionnaire information has been collected, the data is transferred to the system, analyzed by the system, and presented in the form of graphs. In order to protect the privacy of our participants, we have adopted a strictly anonymous form of collection to ensure that all personal information is not disclosed. In addition, we reached out to people of different ages and career paths, and distributed questionnaires using a combination of online and offline to ensure that our data is diverse and that the results are more credible.


    (2) When carrying out popular science activities related to human practice, our team strictly abides by iGEM safety requirements to ensure that all genetic engineering operations are carried out in a strictly controlled laboratory environment. We promise not to take any genetically modified biological GMOs out of the laboratory in order to guarantee the safety of our activities and prevent any form of genetic leakage.

    Learn more in Integrated Human Practices.


    Hardware Security


    Not only do we ensure safety in our experiments, but we also ensure our safety in all aspects of the project, including the hardware. In the hardware section, we have designed a common sugar detection instrument based on lanthanide metal ester fluorescence for sugar detection, and the sugar content in tomato can be known through the fluorescence irradiation intensity. For the detection of the target sweet protein Thaumatin, we designed a rapid quantitative lateral flow assay (LFTA) test strip for Thaumatin, which can quickly detect the content of Thaumatin. (See Hardware for details). In the hardware section, we ensure that no chemicals harmful to the environment are produced during the preparation process, and we ensure that our hardware does not leave the laboratory. In addition, our hardware is an easy-to-use sensor electronics that collects tomato juice samples for fluorescence detection, so it does not pose a hazard to users or the environment.

    smalltitle2



    Conclusion


    "Truly Be A Responsible Scientist or Engineer"


    Finally, we conducted a self-check on the security of our project according to the official requirements of iGEM to ensure that the guidelines of iGEM were followed throughout the process. "Truly being a responsible scientist or engineer" allows our projects to safely move out of the lab and safely into the world.

    Question Answer
    Has the team contributed to biosafety and/or biosecurity? Yes, we contribute to biosecurity. In terms of project preparation and experiments, we have conducted sufficient research to ensure that our chassis, strains and experimental operations will not cause too much harm to the environment; At the same time, we anticipate the possible risks of the project and develop corresponding emergency management measures, so that we can easily respond to them even when danger is imminent.
    Are their contributions well described and/or verified? Yes, our contribution is well described. We visited safety-related departments and asked teachers with different scientific backgrounds to understand the current laws and regulations in an all-round way, described the safety of the project and the laboratory in detail, and wrote specific regulatory legal documents to comprehensively and truly demonstrate our efforts for safety.
    Does the team build on existing knowledge, understanding, tools, or methodologies? Yes, our projects build on existing knowledge, understanding, tools, and methodologies. We use commonly used Agrobacterium for infection, and the strains we use are all scientific backgrounds. In addition, we have obtained the approval of the GMO test field, which can ensure the safe conduct of subsequent experiments.
    In addition to broader security efforts, is the team appropriately managing any risks in the project? Yes, we have anticipated the possible risks in the project and developed measures to manage them. For the dangerous chemical reagents in the experiment, we have developed emergency measures; For the safety issues that may exist on the market of genetically modified foods, we have formulated relevant laws and regulations to supervise. At the same time, we have carried out a comprehensive risk assessment of our projects to ensure that they are safe and reliable.
    Did the team solve the problem of using synthetic biology in the real world? Yes, we have thought deeply about this issue and have done a wide range of practical activities. We take into account the concerns that genetically modified foods pose and the safety of them. To this end, we have established a sound regulatory system to ensure that our projects are safe and reliable in all aspects, and have popularized the advantages and disadvantages of synthetic biology technology to the public, hoping that they can look at it from a more rational perspective.

    louis hide

    Click to Lab Safety
    [1] Joseph JA, Akkermans S, Nimmegeers P, Van Impe JFM. Bioproduction of the Recombinant Sweet Protein Thaumatin: Current State of the Art and Perspectives. Front Microbiol. 2019 Apr 8;10:695. doi: 10.3389/fmicb.2019.00695. PMID: 31024485; PMCID: PMC6463758.
    [2] Yoo SY, Bomblies K, Yoo SK, Yang JW, Choi MS, Lee JS, Weigel D, Ahn JH. The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta. 2005 Jun;221(4):523-30. doi: 10.1007/s00425-004-1466-4. Epub 2005 Jan 29. PMID: 15682278.
    [3] Kurokawa, N., Hirai, T., Takayama, M. et al. An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin. Plant Cell Rep 32, 529–536 (2013). https://doi.org/10.1007/s00299-013-1384-7
    [4] Firsov A, Shaloiko L, Kozlov O, Vinokurov L, Vainstein A, Dolgov S. Purification and characterization of recombinant supersweet protein thaumatin II from tomato fruit. Protein Expr Purif. 2016 Jul;123:1-5. doi: 10.1016/j.pep.2016.03.002. Epub 2016 Mar 8. PMID: 26965414.
    [5] Masuda T, Okubo K, Baba S, Suzuki M, Tani F, Yamasaki M, Mikami B. Structure of thaumatin under acidic conditions: Structural insight into the conformations in lysine residues responsible for maintaining the sweetness after heat-treatment. Food Chem. 2022 Sep 30;389:132996. doi: 10.1016/j.foodchem.2022.132996. Epub 2022 Apr 18. PMID: 35500407.
    [6] Ruiz-Ojeda FJ, Plaza-Díaz J, Sáez-Lara MJ, Gil A. Effects of Sweeteners on the Gut Microbiota: A Review of Experimental Studies and Clinical Trials. Adv Nutr. 2019 Jan.
    [7]EFSA Panel on Food Additives and Flavourings (FAF); Younes M, Aquilina G, Castle L, Engel KH, Fowler P, Frutos Fernandez MJ, Fürst P, Gürtler R, Gundert-Remy U, Husøy T, Manco M, Mennes W, Passamonti S, Moldeus P, Shah R, Waalkens-Berendsen I, Wölfle D, Wright M, Batke M, Boon P, Bruzell E, Chipman J, Crebelli R, Fitzgerald R, Fortes C, Halldorsson T, LeBlanc JC, Lindtner O, Mortensen A, Ntzani E, Wallace H, Civitella C, Horvath Z, Lodi F, Tard A, Vianello G. Re-evaluation of thaumatin (E 957) as food additive. EFSA J. 2021 Nov 30;19(11):e06884. doi: 10.2903/j.efsa.2021.6884. PMID: 34876926; PMCID: PMC8630604.