First of all: this project does not use any animals for experiments.
This project starts from two approaches to experiment:
1. Knock out the branched-chain aminotransferase encoded by the BAT2 gene using intracellular homologous recombination technology, and replace the position of the BAT2 gene with the alcohol acetyltransferase ATF1 gene.
2. On the basis of the first point, the ATF1 gene was placed on the Saccharomyces cerevisiae expression vector and transferred to Saccharomyces cerevisiae to achieve the purpose of reducing the level of higher alcohols in Saccharomyces cerevisiae.
According to previous literature research, we used pHCas9-Nours and pYES2 plasmid vectors, these two plasmids are widely used by scientists without any problems.
We strictly abide by the laboratory's use and management specifications, and there is no risk of leakage.
All of our team members working in the laboratory will be trained in safety and basic laboratory procedures to prevent mishandling such as leaks before conducting experiments.
The laboratory in which we work is equipped with equipment for handling E. coli BL21, which can be protected against leakage by means of protective measures. For example, we will use an autoclave to sterilize all contaminated items, including waste and trash, prior to disposal. All single-use waste is placed in biomedical waste bags. The same goes for waste liquids and bacterial solutions.
Throughout our experiments, we are supervised by professional, class-trained personnel who fully implement the principle of supervision, but not interfering with the experiment. Not only does this ensure that the experiments are safe, but we are able to perform good, efficient experiments.
After the strain was constructed, we performed a growth curve, that is, a test to measure the OD absorbance and simulate alcoholic fermentation. Both tests need to be performed in a sterile environment. Fortunately, our team members are well trained and capable of conducting experiments in a sterile environment.
Any enzymes, strains or antibiotics used in this experiment will not cause any harm to the team members in the laboratory and the ecological environment of the earth, such as destroying the ecological balance.
As long as the training is done properly, the experiments we are involved in do not pose any risk. Thanks to the exploration, development and sacrifice of our predecessors, the experiments we involved have relatively clear steps and procedures, which allow us to have the ability and full control to control the experiments.
During the experimental process, we may deal with some pipette tips and disposable containers like EP tubes. But we all wear lab coats, long pants, and closed-toed shoes, which give our bodies adequate protection against accidents. We also have dedicated needle waste bins that we put in plastic tubes to eliminate this risk factor before disposing of them.
Due to the different impurities of our strains in different winemaking environments, we cannot guarantee that all alcoholic beverages will be safe for certain people, such as those with alcohol allergies. It is extremely unfortunate and disappointing to say that this is beyond our control.
The strains that we constructed contains Nourseothricin and Hygromycin B resistance genes, this is necessary for positive screening.
We did not find any toxic substances produced during the brewing process. Due to the limited equipment of the experimental platform, that means the lab we use, and other factors involved in the experiment let us be forced to can't guarantee that toxic substances will not be produced in other brewing environments that is different from our test environment. In other words, we cannot guarantee the safety of the strains for use in the food industry.
The People's Republic of China has relevant legal constraints on the production and processing of wine.
According to the provisions of Article 2 of the Food Safety Law of the People's Republic of China (2021 Amendment):
The following activities within the territory of the People's Republic of China shall abide by this Law:
(1) Food production and processing (hereinafter referred to as food production), food sales and catering services (hereinafter referred to as food business) ............."
Therefore, the products of this strain are within the scope of this law.
Article 19 of the law stipulates:
"The state implements a licensing system for the production and operation of food. Anyone engaged in food distribution and catering services shall legally obtain a food production license, a food distribution license, and a catering service license. "
Failure to comply will constitute the crime of illegal business operations and be punished according to Article 122 of the law.
Article 26 of the law stipulates that food safety standards should include the following:
"(1) Limits of pathogenic microorganisms in food, food additives, food-related products, pesticide residues, veterinary drug residues, biotoxins, heavy metals and other pollutants, and other substances that endanger human health;
(2) The variety, scope of use and dosage of food additives;
(3) Requirements for the nutritional components of main and complementary foods for infants and other specific groups of people;
(4) Requirements for labels, signs and instructions related to food safety requirements such as hygiene and nutrition;
(5) Hygienic requirements in the process of food production and operation;
(6) Quality requirements related to food safety;
(7) Food inspection methods and regulations related to food safety;
(8) Other contents that need to be formulated as food safety standards."
Failure to comply with this article will result in relative penalties in accordance with Chapter IX of this law, with the most serious being sentenced to life imprisonment.
Based on this, in the scope of legal business, this product is in compliance with the laws of the People's Republic of China. However, the actual situation should be referred to in accordance with the laws and regulations of the sovereign country to which it belongs, such as Iran and other Muslim countries.
Ethically, because we have performed homologous recombination, the product is also a gene editing product, so our product is not the source gene, but because we are out of scientific progress and do not conduct human experiments, according to the European Commission The instrumental of the "Opinion of Ethics of Genome Editing", published on March 19, 2021, is ethically acceptable.
All experimental work of the wet lab were conducted in ATLATL Innovation Center Laboratory, Shanghai, a BSL-1 laboratory.The two images below show the environment of the lab.
Before we started our experiments, we received laboratory safety training and laboratory skills training. All members have been informed of emergency procedures such as fire hydrants, emergency showers, chemical burn treatment and evacuation procedures. We have been given laboratory safety and instruction manuals that we must follow firmly. All of our experimental protocols were written and risk assessed before we conducted our experiments. And during experiments, we pay special attention to experiments that may have risks, and give priority to students with more laboratory experience to give a demonstration to students with less experience. The following laboratory access rules have been formulated:
We will not publish any creatures or products derived from this project, these organisms and products are for research use only. Due to we do not envisage using any animal tissue, the overall risk of our project is relatively low. The entire project will be completed and tested in the laboratory. All wet team members are trained in biosafety prior to entering the laboratory and they are not exposed to biosafety risks. Also, well-trained wet team members are capable of preventing and eliminating these risks.
