Safety regulations serve as the foundational framework for any research endeavor. Despite the prevalence of synthetic biology in engineering microbes, a thorough consideration of safety measures remains imperative. As a result, we meticulously adhered to established safety standards when operating the chassis organisms and reagents for this experiment. Throughout the project, we rigorously maintain adherence to stringent laboratory safety protocols and actively engage safety experts to guide our research endeavors.
Project Introduction
The theme of our project is: "Using synthetic biology to eliminate substances from the air that affect people's lives." On the one hand, because pet waste has a pungent smell, it has a bad effect on our living environment. After consulting the data, we found that the main source of odor is the existence of indole, butyric acid, hydrogen sulfide and other substances. Similarly, we use genetically modified E.coli to give it the ability to degrade these substances and produce aromas, thereby improving household air quality. On the other hand, second-hand smoke will pollute the air environment around us, because it contains many harmful substances, such as nicotine, tar and formaldehyde. Our bodies can also be harmed by long-term exposure to second-hand smoke pollution. So we use genetically modified E.coli to break down substances like nicotine, formaldehyde, and tar, and to break down second-hand smoke in activated carbon.
Our project will focus on the degradation of indole and formaldehyde.
Indole is produced by anaerobic fermentation of food residues (especially proteins) in the gut and is the end product of tryptophan degradation. Ammonia, hydrogen sulfide, volatile fatty acids, phenols, and indole are odor molecules commonly produced in animal farming, among which indole is generally considered to be the most smelly and disgusting odor by human standards, with an olfactory threshold of < 0.0001 ppm[1]. With the development of economic level, people pay more and more attention to the nutrition of pet diet. The rich protein in feed makes indole become the main source of odor in pet excrement.
We selected to introduce indole oxidase ycnE and flavin-dependent monooxygenase FMO into E.coli to construct an engineered E.coli that can degrade indole. GDIAS-5 showed strong indole degradation ability and reached 93.7±5.4% indole degradation rate after 24h culture. Studies have shown that its own ycnE enzyme can achieve the conversion of indole to isatin. In addition, E.coli itself is also the source of indole, and its own expression of tnaA enzyme can convert tryptophan into indole, which is also the main source of odor ofE.coli. Therefore, we used the λ red homologous recombination system to eliminate the tnaA enzyme gene of E.coli itself to construct odorless E.coli, and conducted further gene editing to introduce genes encoding tnaA enzyme and FMO enzyme, so as to eliminate the odor of E.coli itself and achieve exogenous indole degradation.
Formaldehyde is a harmful gas with strong irritating and choking odor and is classified as a primary carcinogen[2]. We made E.coliexpress hexose 6-phosphate synthase (Hps) and 6-phospho3-hexose isomerase (Phi) genes from Bacillus methanesticus to establish the ribulose monophosphate cycle and achieve formaldehyde assimilation. The frmA gene encodes the glutathione-dependent formaldehyde dehydrogenase in E.coli, which converts formaldehyde to formic acid. By knocking out frmA gene, formaldehyde can be prevented from producing carbon dioxide through the dissimilation pathway, and formaldehyde can be converted into substances that can be utilized by E.coli through the assimilation pathway.
Under the guidance of teachers and the continuous efforts of teammates, our ideas will be born.
Laboratory Safety
Laboratory Risk Assessment
As this experiment primarily falls within the realm of synthetic biology, a subset of genetic engineering, it inherently encompasses considerations related to gene safety. Hence, in preparation for the experimental procedures, we conducted an exhaustive review of the 'Genetic Engineering Safety Management Measures'[3] mandated by the Central People's Government of the People's Republic of China. We pledge to execute the experiment with unwavering adherence to these regulations to guarantee the safety and reliability of our experimental procedures.
In addition, we will use non-toxic E.coli (Top 10) as the chassis organism for this experiment project. E.coli is a strain listed on iGEM's official website, so it has high safety and recognition, low pathogenicity, and no public health risk. According to the "pathogenic microbiology laboratory Biosafety management Regulations", E.coli belongs to the third class of pathogenic microorganisms, that is, can cause human or animal diseases, but under normal circumstances do not pose serious harm to people, animals or the environment, the risk of transmission is limited, rarely cause serious diseases after laboratory infection, and have effective treatment and prevention measures of microorganisms.
Finally, we evaluate the safety level of the laboratory, and the evaluation result is: a biosafety level laboratory. Our laboratory is a basic laboratory, often for basic teaching and research laboratories, dealing with risk level 1 microorganisms.
To sum up, the experimental project and the laboratory have a high safety factor.
Experimental Operation Training
At the beginning of iGEM, we actively carried out training activities on experimental safety and standardized operation.
On July 10, 2023, all members of the experimental team, led by advisor, Wang Xingjie, received biosafety training and general and chemical safety training, which covered safety-related concepts, basic emergency procedures, and how to handle microorganisms. In this way, we ensured the safety of our researchers throughout the project.
Laboratory Requirements
(1) laboratory entry disinfection, not eat in the laboratory, allowed to drink beverages, if you want to eat, please eat outside the laboratory or the canteen;
(2) Make a plan before the experiment, conceive the process well, it is best to write it down to reduce the probability of error;
(3) The experiment should be carefully conducted, strictly in accordance with the operating procedures, and pay attention to changing the habits of life;
(4) Super clean and light box first wear gloves to disinfect before operation, to prevent contamination of other bacteria;
(5) The experimental operation must wear a lab coat, mask, and gloves;
(6) After the experiment is completed, the experimental platform must be cleaned, the instrument should be placed in the designated place, the pipette should be adjusted to the maximum range, the waste liquid cylinder should be dumped and washed, and the power supply of the instrument should be turned off.
Dangerous Reagent Management and Use Method
Although our project involves toxic substances such as nicotine, benzo[a]pyrene, formaldehyde, indole, hydrogen sulfide, etc., they are all used in very low concentrations. Due to very low concentrations, they are greatly reduced in toxicity and carcinogenicity and are considered safe for individuals and the environment.
Any operation involving fungi and toxic reagents, we will operate inside the ultra-clean workbench, and strictly wear masks and gloves to prevent toxic reagents from contact with the human body.
For protein validation such as indole, formaldehyde and butyric acid, we perform polyacrylamide gel electrophoresis. In this process, the eluent and buffer used in our configuration are toxic reagents. In order to ensure the safety of the experimental personnel, we will wear double gloves and carry out in the fume hood.
In addition, when we illuminate the gel with ultraviolet lamps to observe the bands, in order to reduce the damage of ultraviolet rays, we will wear goggles to protect our eyes.
In addition, any bacterial stocks and laboratory waste were strictly autoclaved before discarding them to ensure that they are not released into the environment and pose a threat to human safety. If accidentally released, it poses no threat to human health due to the good characteristics of the bacteria we use, such as non-virulence and non-pathogenicity.
| Substance Name | LD50 | Use Concentration | Whether it is safe | |||
| Nicotine | 25mg/kg | 10 mg/kg | Y | |||
| Formaldehyde | 0.1mg/m3 : pharyngeal discomfort 0.5mg/m3 : Irritates the eyes and causes tears 0.6mg/m3 : causes sore throat, nausea, vomiting, pulmonary edema, etc 30mg/m3 : causes death | 0.05 mg/m3 | Y | |||
| benzopyrene | 500mg/kg | 378 mg/kg | Y | |||
| indole | 1000mg/kg (oral administration) | 580mg/kg | Y | |||
| Butyric acid | 5000-10000 mg/kg | 4550mg/kg | Y | |||
| Hydrogen sulfide | 100300ppm | 150ppm | Y | |||
| Ammonia | 900 ppm | 270 ppm | Y | |||
| DNA nucleic acid dye | / | Safe concentration and wearing double gloves | Y | |||
| Dye solution | Isopropanol | 2524mg/kg | 256mg/kg | Y | ||
| Commassie Blue Fast Staining Solution | / | Safe concentration, fume hood experiment | Y | |||
| Destaining solution | glacial acetic acid | 1060mg/kg | 268mg/kg | Y | ||
| methyl alcohol | 30ml | Safe concentration, fume hood experiment | Y | |||
In case of accidental ingestion of the above reagents, the emergency treatment is as follows:
(1) Skin contact: Remove contaminated clothing and rinse with plenty of running water.
(2) Eye contact: Lift eyelids, rinse with running water or normal saline and seek medical attention promptly.
(3) Inhalation: Quickly leave the scene to a place with fresh air to keep the respiratory tract unobstructed. If breathing is difficult, give oxygen; If breathing stops, immediately give artificial respiration and seek medical attention.
(4) Ingestion: Drink enough warm water to induce vomiting, lavage the stomach, induce ejaculation and seek medical attention.
Experimental Parts
The biological parts we use and construct do not raise any safety concerns. They do not acquire any disease-causing mechanisms that could cause harm to human health.
According to WHO, if the organism is a pathogen that can cause disease in humans or
animals, but is unlikely to cause serious harm to laboratory workers, the community, livestock, or the environment, it is classified as risk 2, which means moderate individual risk, low community risk. While there is a risk of infection with the use of these parts in the laboratory, the risk of infection transmission is very small with effective treatment and prevention measures.
Kill Switch
For biosafety reasons, we refer to a suicide device designed by TU Darmastadt's team to help us accomplish the self-destruction of E.coli in this experiment.
At the heart of the device is the use of E.coli 's hokD gene, whose overexpression disrupts cell wall function and leads to cell death. It has been shown that the hokD gene can be used to design effective suicide functions. To avoid cell death due to basal expression of hokD, the project designed hokD to be controlled by the T3 promoter (phi4.3), but the expression of the T3 polymerase itself is controlled by the pBad promoter, which is inhibited by AraC. In the presence of glucose, the AraC repressor tightly inhibits the expression of T3-polymerase, so that the expression of hokD is inhibited and the bacteria can grow at a normal growth rate under controlled conditions. When released into the environment, the glucose is depleted and the T3 promoter causes the expression of the hokD gene and initiates the bacterial white kill response.
