In order to ensure that our patch containing genetically engineered organisms can be safely applied in real circumstances, we evaluated the material of the patch to prevent the leakage of E. coli and designed a system of biosafety.
The material of the patch mainly consists of bacterial cellulose (BC) and filter membranes. The pore of BC is 10- 12 nanometers, which is less than the size of E. coli. In this way, the engineered bacteria will be constrained in two membranes of the patch, avoiding its leakage throughout the usage.
After ensuring the E. coli will not leak into the environment, it becomes imperative to take action and completely eradicate genetically engineered bacteria to mitigate any potential risks. One approach is to introduce a "suicide gene" into E. coli, which compromises its biological function. However, it can be challenging to ensure that E. coli remains viable during treatment and terminates promptly post-treatment. In addition, expressing "suicide genes" is a waste of energy for engineered strains. The production process consumes a lot of energy and material processes, which reduces the efficiency of the bacterial function. Another option previously considered was modifying the environment to eradicate these engineered bacteria. Increasing temperature can cause E. coli's proteins to denature and its membrane lipids to melt, resulting in cell death. Thus, a high-temperature approach may be a viable solution for eliminating residual E. coli in used patches.
In China, hotpot cuisine holds a distinctive position, characterized by the presence of a boiling broth pot at the dining table where patrons can cook raw ingredients. Recently, a novel culinary innovation known as the 'Self-Heating Pot' has emerged. This innovative cooking apparatus employs a chemical reaction to generate heat, thereby mimicking the conventional hotpot's functionality. Inspired by this concept, our research endeavours sought to apply a similar principle in the context of waste management. Specifically, we aimed to design an advanced biosafety system that harnesses the exothermic reaction occurring between quicklime and water to efficiently generate thermal energy for waste disposal.
According to the World Health Organization, the majority of E. coli are killed when the temperature exceeds 65°C. With this in mind, we intend to employ the exothermic reaction between quicklime and water to generate heat for high-temperature treatment. During the design of this principle, we recognized that the product of this reaction, calcium hydroxide, exhibits strong alkalinity. Consequently, if patches are immersed in this solution, E. coli might also succumb to the destructive effects of the alkaline environment on their cell membranes. Based on these insights, we undertook a preliminary experimental design.
After determining the temperature, we model the appropriate amount of quicklime and water for our biosafety system.. One way to represent this reaction is by using a chemical equation: CaO+ H2O=Ca(OH)2. Upon analyzing the data, it has been found that under standard conditions, the enthalpy change for producing calcium oxide is -635kJ/mol, whereas for water, it is -286kJ/mol. Furthermore, the enthalpy change for producing calcium hydroxide is -985kJ/mol. Utilizing this information, it can be calculated that the reaction between 1mol of quicklime and 1mol of water results in the production of 64kJ of heat energy.
During the development of an efficient biosafety device, it's crucial to precisely determine its geometric design as well as the exact amounts of quicklime (CaO) and water required for its functionality. We've designed the size of the biosafety device to approximately be 12cm x 6cm x 8cm. For the packaging material, we opted for heat-resistant polyethene with a design thickness of 1mm. As for the initial and ambient temperature settings, we set them both at 25°C. According to relative research, we've determined the thermal conductivity of polyethene to be 0.33W/m*K.
For modelling purposes, we made several assumptions: We neglected any time-dependent changes in the specific heat capacity of the solution, assuming the entire solution's specific heat capacity to be equivalent to that of water, which is 4186J/kg*K. Additionally, we assumed the reaction to be of zero order, meaning its rate doesn't vary with time. Due to the lack of experimental simulation, we roughly set the reaction rate at 0.0008kg/s, which falls within a reasonable range for reaction rates.
Based on our modelling results, we observed that the internal temperature of the device can be maintained above 65°C for 29.5 seconds. At this time, 100 millilitres of water was added inside the device, and the quicklime inside the heating pack weighed 40 grams.
Our product's design seamlessly amalgamates two primary functions: the storage of patches and their subsequent biosafety. Such an integrated approach is not merely an aesthetic choice but a strategic one, aiming to maximize space utilization and concomitantly reduce associated costs. The design dictates that the utilized patches be deposited into the heating compartment situated on the lower tier. Once the patches have been expended, the heating mechanism can be activated. Concurrently, water is introduced into the lower-tier device. As the water level surpasses the demarcated scale line, the device initiates the heating process, ensuring effective processing of the used patches. During the treatment process, it is also necessary to design some exhaust holes, which can effectively avoid the risk of explosion inside the device. Post this treatment, the entire containment can be safely disposed of in a conventional waste bin. This ensures that our patches, post-usage, do not contribute to environmental degradation or contamination.
We conducted random interviews with dozens of iGEM teams over the past few years. Our findings revealed that 86% of the teams have considered biosafety, and 94% of team members are familiar with the transformation of Streptococcus pneumoniae. However, not a single team has taken into consideration how to prevent horizontal gene transfer from deceased bacteria, such as in the case of Streptococcus pneumoniae transformation experiments.
However, during the design process, we recognized certain limitations in our product. When designing the patch, we were reminded of Frederick Griffith's transformation experiment with pneumococcus. Griffith's experiment demonstrated that a non-pathogenic strain of bacteria could be made pathogenic by exposure to heat-killed pathogenic bacteria, thus proposing the existence of a "transforming principle" that carried genetic information. Consequently, while we could achieve basic inactivation of the engineered bacteria during high-temperature treatment, we still could not ensure the prevention of horizontal gene transfer from the bacterial DNA. The most effective measure to inhibit horizontal gene transfer is to carbonize the DNA at extremely high temperatures; only in this way can we ensure that the genes of the engineered bacteria will not contaminate the external environment.
Therefore, we employed a muffle furnace to subject our engineered bacteria to high-temperature treatment, demonstrating that this heat-based approach can effectively degrade DNA.
In LB medium, add 100μg/ml ampicillin. Inoculate the bacteria E. coli Rosetta/pET23b-hEGF and incubate on a shaker set at 220 rpm and 37°C until the OD600 reaches approximately 1.0. Collect 100mL of the bacterial culture and centrifuge at 4°C and 10,000 rpm for 1 minute to obtain the bacterial pellet. For the control sample, extract DNA using the bacterial DNA extraction kit (Tiangen, China). Place the bacterial pellet into a mortar. Preheat the muffle furnace to 200°C. Place the mortar inside the muffle furnace and heat for 1 hour. After the treatment is completed, carefully remove the mortar from the furnace using tongs. Place the mortar on an ultraclean bench that has been pre-treated with ultraviolet light for natural cooling. Ensure the fan of the ultraclean bench is turned off to prevent the sample from being dispersed. Resuspend the sample treated in the muffle furnace using 100μl of DNA buffer. Subsequently, perform PCR reactions on both the control and treated samples to amplify the hEGF gene. Then we verify the PCR results using agarose gel electrophoresis. The result is shown in the figure.
Yet, it must be acknowledged that with the current methods, we cannot achieve the carbonisation of DNA solely through simple, low-cost, and home-operable means. Therefore, we still need to explore more effective, safer, and practical treatment measures.
At the same time, we have also done some related activities to call more attention to the problem of horizontal transfer that may occur in synthetic biology.Here are a few succinct activity descriptions and their outcomes:
Workshops on Horizontal Gene Transfer: Conducted informative workshops to raise awareness among teams about the potential risks of horizontal gene transfer in synthetic biology.
Outcome: Increased teams' understanding of biosecurity concerns.
Biosecurity Challenge: Organized a biosecurity challenge to promote innovative solutions for preventing horizontal gene transfer.
Outcome: Sparked creative thinking and problem-solving in the iGEM community.
Interactive Discussions: Facilitated interactive discussions on biosecurity issues during team meetups and events.
Outcome: Fostered a culture of dialogue and collaboration on biosecurity matters.
Best Practices Handbook: Developed a comprehensive handbook outlining biosecurity best practices and distributed it to participating teams.
Outcome: Provided teams with valuable guidelines to improve their biosecurity efforts.
the safe chassis microorganism
We have selected E. coli Nissle 1917 (EcN 1917) as our chassis microorganism. This choice is grounded in over a century of research since its confirmation in 1917 as a microorganism with high antagonistic activity within the intestinal tract. EcN 1917 has consistently proven itself as a robust and effective microorganism and is widely recognized as a non-pathogenic member of the Escherichia coli family. This recognition primarily stems from the fact that this strain lacks pathogenic factors, does not produce any cytotoxins, is non-invasive, and can be rapidly neutralized by non-specific host defense mechanisms (Sonnenborn & Schulze, 2009). Furthermore, it does not possess any known pathogenic characteristics (Sonnenborn, 2016). These conclusions are based on the characterization of the complete plasmid (Blum-Oehler et al., 2003) and genomic DNA sequences (Reister et al., 2014) of this well-known E. coli strain. These studies have provided substantial evidence regarding the safety of E. coli in medical applications.
Based on this extensive research foundation, Mutaflor, a therapeutic drug containing the active component EcN 1917, has been authorized for sale in multiple countries, including Germany, for nearly a decade for the treatment of intestinal dysbiosis (Behnsen et al., 2013).
Given these advantages, EcN 1917 has found wide application as a chassis microorganism in synthetic biology and rapidly become one of the most popular and extensively studied chassis microorganisms (Lynch et al., 2022). Moreover, since EcN's genetics are traceable, the endogenous EcN plasmids can be genetically manipulated and stably maintained, further ensuring the safety of this strain in synthetic biology applications (ACS Synth. Biol., 2021).
the consideration of gene
We have selected FadL, FadD, and hEGF as the core genes for our product. Our research indicates that these three genes exhibit stable expression levels, do not produce toxic factors, show no long-term side effects, and have no impact on the environment. Therefore, they are suitable for use as gene fragments. Additionally, we have observed that these three genes have been widely utilized in experiments, and there have been no reported safety concerns associated with their use.
the safe engineering approach
Substrate: Long-Chain Fatty Acids (LCFA)
Product: Epidermal Growth Factor (EGF)
Our substrate is Long-Chain Fatty Acids (LCFA), a type of fat commonly found in the food, cosmetics, and pharmaceutical industries. It is easily degradable and poses no toxicity to the environment or the human body.
Ultimately, our engineered bacteria will produce Epidermal Growth Factor (EGF), a peptide that promotes epidermal healing. This peptide needs to bind with the Epidermal Growth Factor Receptor (EGFR) to exert its function and does not bind with other receptors. According to current research, there is no evidence to suggest that EGF is cytotoxic, and there have been very few reports of severe allergic reactions or EGF-related cancers in clinical medicine.
Furthermore, we have noted that EGF is widely used and well-received in fields such as cosmetics and medicine, which attests to the safety of EGF.
Promotion of product
We have conducted potential customer interviews and questionnaire surveys, and the results are as follows. In general, patients are willing to accept it, but there are some concerns, which we think is related to the novelty of our technology. In order to further understand the user's acceptance of the treatment of modified E. coli, we issued an online questionnaire. The use of E. coli repatch was introduced. The survey shows that 52.94% of users are willing to use drugs, 47.06% of users are not willing. The main concerns of most users are safety and lack of understanding of E. coli treatment. We believe that as we continue to improve the patch and do biosecurity, more and more people will be willing to try it.
Making Poster of Historical Lab Accidents For Education
Many people think that laboratory safety rules are irrelevant or unnecessary and should not be taken seriously. The result is that they often make mistakes during the experiment due to improper operation, resulting in huge security risks. In order to reduce lab operating accidents, our team decided to use tragic accidents that happened in labs before to caution people about the necessity of observing experimental rules and being more careful during experiments. We chose three stories, including the death of professor at Dartmouth College Karen E. Wetterhahn due to contact with dimethylmercury solution, Cecil Kelly in Los Alamos National Laboratory who was killed by a deadly dose of neutrons and gamma rays, and the Marburg Virus pandemic originated from monkeys for polio research. We made a poster based on the three stories and gave some advice on how to implement laboratory safety implementation. The poster was stuck onto the lab walls of The High School Affiliated to Renmin University of China, China Agricultural University and other high school and universities in Beijing, so students could be alarmed by real-life accidents and pay more attention during lab experiments.
Ensuring the safety in laboratory is an important aspect and a responsibility that RDFZ-CHINA is committed to. At iGEM 2023, we have taken several measures to create a secure laboratory working enviroment for our team members when doing the research and experiments.
General Lab Training
Considering the importance of laboratory safety, we provide the standard training of experimental procedures and common operation rules for each team member before they enter the lab. It includes the laboratory safety regulations, methods for the safe use of laboratory facilities, methods for handling emergency situations, storage of hazardous materials and drugs in laboratories, biosafety, explosion and fire prevention. In addition, each student is given a requirement to finish reading
Safety Regulations
1. Any foods and drinks are forbidden in the lab.
2. Everyone in the lab should wear gloves and lab coat.
3. Everyone should follow the tips in the equipment when use them.
4. Everything should be sterilized with alcohol before they are put in super clean bench.
5. Do not speak when you are facing somewhere that should be without any other microorganism (e.g., super clean bench, incubator and refrigerator).
6.The biological laboratory shall conduct the necessary risk assessment and establish risk control procedures
7.Biological laboratories shall comply with the national standard "General Requirements for Laboratory Biosafety GB19489-2008" BSL-1~BSL-4 terms of this experiment
8.The room should be reasonably designed, and all facilities, equipment and materials (including protective barriers) should meet the relevant national standards and requirements.
9.The staff of biological laboratories shall voluntarily engage in laboratory work on the premise of foreknowing the potential dangers of experiments. All laboratory policies, regulations and procedures must be followed. Must go through safety education and professional training and assessment qualified, in the independent work should also be under the guidance of senior experimental technical personnel training, to meet the qualified standards before you can start to work.
10.In addition, animal testing staff should also hold a state-recognized qualification certificate.
11.The staff of BSL-3 and BSL-4 biological laboratories must keep background serum for relevant testing before starting work, take regular physical examinations and establish health records. If there is a vaccine, it must be taken.
12.There shall be more than two staff members engaged in highly pathogenic microorganisms related experimental activities.
13.Biological laboratories shall establish experimental files to record the use of the laboratory, safety supervision and the whole process of biological hazards from entering the laboratory to the final destruction. The retention period of experimental files engaged in experimental activities related to highly pathogenic microorganisms shall not be less than 20 years.
14.Collection of highly pathogenic microorganisms should be carried out in equipment with corresponding safety protection level, the collection process must strictly prevent the spread and infection of pathogenic microorganisms, and the source of samples, collection process and method should be recorded in detail. Its transportation shall comply with the requirements of corresponding national regulations and standards.
15.The laboratory of highly pathogenic microorganisms shall take effective security measures to strictly prevent highly pathogenic microorganisms from being stolen, robbed, lost, and leaked, so as to ensure the safety of the laboratory and pathogenic microorganisms. If highly pathogenic microorganisms are stolen, robbed, lost, or leaked from the laboratory, they shall immediately report to the school, the local health authorities and the public security Bureau.
Reference：Tsinghua University Biological Laboratory Safety guidelines 2019 edition
Virtual Training for Lab
Apart from general safety training, we adopted a new manner of education: the virtual experiment. It uses virtual software on the computer to simulate the effects of doing experiments. We created accounts for each team member to do the practices, providing their chances to experience the operation. The virtual program includes an explanation of key points, questions, and special tips. Our team members will go through all the procedures and pass the exam to be qualified to get involved in the laboratory. The most significant benefit of this program is the repeated practice. Through a good program design of the simulation experiment platform, the drawbacks of limiting students to only perform "correct" operations in the past are greatly improved by allowing students to select the "wrong" one. In this way, educators can use the virtuality of simulation experiments to simulate many dangerous situations that can not be borne in real experiments, so that students can immerse themselves in the danger of the laboratory. Taking advantage of nearly unlimited opportunities for trial and error cultivates the self-exploration to firmly remember dangerous situations, their causes, and how to deal with them. At the same time, it can help users understand a variety of drugs, equipment, and their safety hazards, truly becoming a qualified safety operator.
Graphic Tips
In addition to training outside the lab, we made stickers in which researcher uses different equipments and paste it in the lab as graphic tips, including pipette, aseptic handling box, centrifungal machine. It could remind our teammates how to properly operate and attach importance to essential details. In order to be more ideal, we shot a video that shows the specific and standard operation upon each devices, demonstrating through QR codes which is printed on the corresponding drawings. In this way, if team members are not familiar or not sure with the operation, they can scan the code to take a look of it. We still design cartoons for the using of lab equipment, which is cooperated with THINK-CHINA. In the cartoon, the operations are drawed one scene by one scene. Knowing of lab rules are so improtant to keep students from emergency or hurting. We must ensure that everyone knows how to protect themselves.
Sonnenborn, U., & Schulze, J. (2009). The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic. Microbial Ecology in Health and Disease, 21(3-4), 122–158.
Sonnenborn, U. (2016). Escherichia coli strain Nissle 1917—from bench to bedside and back: history of a special Escherichia coli strain with probiotic properties. FEMS Microbiology Letters, 363(19), fnw212.
Blum-Oehler, G., Oswald, S., Eiteljörge, K., et al. (2003). Development of strain-specific DNA reactions for the detection of the probiotic Escherichia coli strain Nissle 1917 in fecal samples. Res Microbiol, 154, 59-66.
Reister, M., Hoffmeier, K., Krezdorn, N., et al. (2014). Complete genome sequence of the Gram-negative probiotic Escherichia coli strain Nissle 1917. J Biotechnol, 187, 106-7.
Behnsen, J., Deriu, E., Sassone-Corsi, M., et al. (2013). Probiotics: properties, examples, and specific applications. Cold Spring Harb Perspect Med, 3, a010074.
Lynch, J. P., Goers, L., & Lesser, CF. (2022). Emerging strategies for engineering Escherichia coli Nissle 1917-based therapeutics. Trends Pharmacol Sci, 43(9), 772-786.
ACS Synth. Biol. (2021). Plasmid vectors for in vivo selection-free use with the probiotic E. coli Nissle 1917, 10, 94-106.