Safety is not only a demonstration of the team's sense of responsibility, but also the basic guarantee for the development of the project. Guided by the spirit of iGEM, our team chose to explore the microbial synthesis of the green pesticide 5-ALA, and in the process, strictly adhered to various safety regulations, identified and avoided related risks. We spared no efforts to minimize the possible harm caused by the project to team members, other members of the laboratory and society.
Our team always keeps the iGEM safety standard in mind, and actively participates in safety training activities while mastering basic laboratory operation skills. In view of the fact that our project involves technologies such as CRISPR and droplet microfluidic high-throughput screening, we have noticed the importance of ensuring the safety of the project, and have thought about countermeasures in advance and reflected on ourselves in time during the development of the project. It is worth mentioning that we also discussed the potential risks of the project and our contribution to food security. In short, we refer to the relevant laboratory safety policies of the country and the university, carry out the project in strict accordance with the highest safety standards of the laboratory, and do our best to make our contribution to the safety of synthetic biology and product application while determining the safety of the project.
1. Safety facilities in the laboratory
The experiment was carried out in the LICME laboratory of the School of Food and Pharmaceutical Engineering of Nanjing Normal University, and our laboratory belongs to the Level 1 - Standard Microbiology Laboratory, which complies with the Regulations on the Biosafety Management of People's Republic of China Pathogenic Microorganisms (https://www.gov.cn/gongbao/content/2019/content_5468882.htm) and the Safety Code for Laboratories in Colleges and Universities (https://www.gov.cn/zhengce/zhengceku/2023-02/21/5742498/files/493d9b7d76014c50903cbc16e09c2253.pdf).
All of our laboratory activities comply with the "Nanjing Normal University Laboratory Safety Management Regulations" (ssc.njnu.edu.cn/info/1024/1629.htm). The laboratory has a clear and orderly functional partition to ensure the effectiveness of the laboratory. Next to equipment that may be dangerous due to improper operation or other reasons, such as autoclaves and gel electrophoresis machines, we post safety signs and precautions and implement use registration. In addition, in addition to necessary safety measures such as goggles and protective gloves, we are also equipped with emergency equipment such as first aid kits, sprinklers, and eyewashes to fully ensure the safety of laboratory personnel.
Safety features in our labs include:
(5) Autoclaves and other sterilization equipment;
(7) Fire-fighting equipment and first aid kits;
(8) Emergency lighting device.
2. Safety training for personnel
Before the start of the project, all members received comprehensive laboratory safety and skills training to ensure that the team members can establish the concept of "safety is paramount", and have a deep understanding of the laboratory safety management system and the correct and standardized use of various instruments in the laboratory. At least one PI or Instructor is present during our experiments to ensure the safety of the project and to provide guidance at the right time.
(1) Overall safety
The project members all have a certain foundation of experimental skills, and have systematically studied and passed the course "Introduction to Safety Engineering", so that they have a full understanding of the risks of working in a laboratory environment, and are familiar with the steps to mitigate and deal with potential accidents.
(2) iGEM security training
Before the start of the project, Prof. Wang Yuetong and Prof. Shi Tianqiong once again carried out safety training for us for this project, including plasmid extraction and transformation, gel electrophoresis, peristaltic pump, microscope operation and other experimental skills training, and careful registration of experimental equipment use, proper disposal of waste and other necessary laboratory safety precautions. In addition, the laboratory has strict safety management norms, and anyone who ignores laboratory safety and abuses the instrument will be restricted from using experimental equipment.
By implementing these training measures, we maximize the safety and reliability of the experiments, thereby reducing the potential risks of the project to team members and the external environment.
3. Laboratory waste disposal
In the laboratory, first of all, we need to classify and place all wastes, including but not limited to ordinary garbage, solid waste, organic waste liquid, inorganic waste liquid, etc., and then carry out preliminary treatment according to the different properties of the wastes to ensure that they will not cause harm to the laboratory environment and personnel. For example, sharp objects such as used toothpicks should be disposed of separately in a specific trash can; After the experiment, the equipment should be thoroughly cleaned and inspected to prevent the presence of residues and bacteria; For the bacteria that are too old, the bacteria should be sterilized at high temperature and then poured into the organic waste tank; In addition, our laboratory processes a batch of solid waste on a weekly basis on a regular basis. These measures further enhance the safety of our projects.
1. Genetic safety
Before constructing the plasmid, we check the source of the gene we want to use. They are all from the whitelist provided by iGEM.
| Part's name | Risk associated |
|---|---|
| RChemA-ALAS | Derived from Rhodobacterium capsulatum No hazardous effect |
| AFhemA-ALAS | Derived from Agrobacterium No hazardous effect |
| RPhemA-ALAS | Derived from Rhodopseudomonas palustris No hazardous effect |
Our project involves CRISPR transposable and CRISPR inhibition, and a piece of guide RNA (none of the target genes in this project) targeted at the target gene is used in the CRISPR system, which is also whitelisted.
2. The strain is safe
The E. coli BL21 and DH5a we used are all in risk group 1 in the white list, these strains are not pathogenic, the risk to humans and the environment is extremely low, they are widely used in genetic engineering, protein expression, metabolic engineering and other fields, have mature culture and operation methods, and are sensitive to conventional disinfection and sterilization methods. During the operation of the project, we strictly adhere to the laboratory practice and take the following measures to ensure safety:
3. Microfluidic high-throughput screening related experiments are safe
(1) Chip security
In this project, we independently designed the chip channel and entrusted Suzhou SMIC Qiheng Co., Ltd. to process and manufacture PDMS chips. Water-in-oil droplets were generated in the chip by flow focusing method, and the oil phase was 2% fluorinated surfactant 008-FluoroSurfactant, and the water phase was E. coli solution containing mutagenesis. Polydimethylsiloxane (PDMS) is considered the material of choice for the manufacture of microfluidic chips and offers a number of advantages:
(1) Easy to bond, can be combined with itself or other materials, easy to manufacture;
(2) Easy to observe under a microscope;
(3) Non-toxic and good biocompatibility;
(4) Good light transmittance and strong permeability of gases (oxygen, carbon dioxide, etc.);
However, it also leads to some problems: incompatibility with organic solvents, and the inability to support certain quantitative experiments due to three effects, such as small hydrophobic molecules being absorbed by the channel wall, biomolecules being adsorbed by the channel wall, and water evaporating through the channel wall to change the concentration of the solution, etc. We try to introduce various modification strategies to overcome these shortcomings as much as possible, such as increasing the flow rate of the inner and outer phases to allow the fluid to pass through the channel quickly, and the inlet and outlet are closed by PE pipes to prevent evaporation.
(2) Droplet safety
Through parallelization experiments, we have explored the concentration of the inner phase bacterial solution, the type of oil phase, the type and concentration of surfactant, the velocity of the initial fluid, and the calculation of the single package rate of the droplet. By consulting a large number of literatures, it was found that the main oil phases used were fluorinated oil, mineral oil, and paraffin oil; The main surfactants used are Tween 80, span80, fluorinated surfactant, EM90, etc., and the most suitable oil phase and surfactant are fluorinated surfactant and fluorinated oil through experiments.
The advantages of fluorinated oils are:
(2) Low viscosity, good gas solubility;
(3) It can stably generate 1~300 μm droplets, and the droplet generation frequency is high;
(4) Suitable for cell culture experiments;
But the disadvantages are:
(1) It is volatile and has poor thermal stability;
(3) Surfactants need to be added.
Due to the characteristics of surfactants, the prepared fluorinated oil needs to be stored at low temperature and taken at any time.
(3) Mutagenic safety
ARTP stands for Atmospheric and Room Temperature Plasma, and is capable of generating plasma jets with a high concentration of active particles (including helium atoms, oxygen atoms, nitrogen atoms, OH radicals, etc. in the excited state) at atmospheric pressure with temperatures ranging from 25-40 °C. The plasma jets can also be used to produce a high concentration of active particles (including helium atoms, oxygen atoms, nitrogen atoms, and OH radicals in the excited state).
The application of ARTP in the biological field has the following advantages:
(1) ARTP has low temperature, high concentration and variety of active particles;
(3) ARTP equipment is simple, easy to operate, low operating costs;
(4) ARTP is environmentally friendly, causing no pollution or harm.
4. Product condition
(1) Our laboratory has a complete safety management system, we strictly follow the waste disposal regulations of the laboratory, and properly dispose of the waste generated in the experiment to ensure that our products or other products with potential safety hazards will not spread to the outside world at will.
(2) Our product, 5-aminolevulinic acid (5-ALA), is a non-toxic, environmentally friendly non-protein-derived amino acid, which can be degraded under natural conditions and does not leave harmful residues. It will not cause harm to the environment or human body.
5. Predict future risks
We believe that in the future further product production process, more green, environmentally friendly and sustainable production technologies should be adopted to avoid the pollution and safety problems that may be caused by chemical synthesis and bioengineering. In addition, the dosage and method of use of 5-ALA for different crops should also be clarified to prevent possible safety problems during use.
Pesticides are widely used to protect food production and meet global food needs, and their use effectively suppresses crop diseases and pests and ensures crop yields.
At present, the global pesticide market is dominated by chemical pesticides, which have a rapid effect, remarkable effects, and a wide range of use, and are widely used. However, the persistence of chemical pesticides in the soil, pest resistance, and the impact on biological health and the environment cannot be ignored, and the unsafety of chemical pesticides has also become an important problem in agricultural production.
In this context, our team proposed a strategy to produce the biopesticide 5-ALA using synthetic biology methods.
We hope to use the safety of biopesticides themselves, that is, they are gentle to the environment, have target specificity, have less residue in the soil, and have little impact on surrounding organisms, combined with synthetic biology technology, to achieve high yield of biopesticides and reduce the use of chemical pesticides. Truly realize the effective guarantee of food security while protecting the safety of the environment and organisms.
Prior to the start of the project, the team members carefully studied and reviewed the following documents and policies to ensure that our project content complies with the relevant policies and regulations:
(1) People's Republic of China Food Security Law (http://www.icama.org.cn/zwb/detail/27522)
(2) Administrative Measures for the Safety Evaluation of Agricultural Genetically Modified Organisms（http://www.icama.org.cn/zwb/detail/27518）
