Our project is as reliant on genetic engineering, which modifies the gene beyond the limits of natural evolution, as any other projects related to synthetic biology. Alternation of genes can provide opportunities and benefit all human kinds.However, it is also likely to pose a threat to human beings and the environment we live in. Thus, assessment on biosafety and managing our GMOs is crucial for our project and to help us with that we interviewed molecular biology experts on transgenic safety and the potential risks. https://2021.igem.org/Team:AISSU_Union/Integrated_HP
In terms of microbes,we chose the E.coli (DH5α) and the yeast Saccharomyces cerevisiae (BY4741) for early laboratory studies and testing.DH5α is the most frequently used model organism for high copy number plasmid amplification and Saccharomyces cerevisiae is widely used due to its efficient homologous recombination mechanism and fermentation capabilities. The researchers have comprehensively studied on both strains and used them extensively in various experiments. Moreover, E.coli (DH5α) and Saccharomyces cerevisiae are listed on the IGEM white list, meaning that they do not require additional registrations.
Genetic engineering and plasmid construction
Our project is highly dependent on genetic engineering and the various problems related to biosafety may arise from artificial alternation in genes. As a result, any modification plan is carefully assessed on its safety and necessity before implementation.
We plan to construct two plasmids that are responsible for 2 types of metabolism pathway and the main techniques used are Golden Gate Assembly, Gibson Assembly, Restriction endonuclease ligation and Homologous Recombination for yeast. The techniques are used on a large scale because of their convenience and efficiency. Please check out the result section to see more detailed information about construction result of related plasmids. (https://2021.igem.org/Team:AISSU_Union/Design)
We put possibility of accidental release into consideration when we chose our strains, so Saccharomyces cerevisiae (BY4741) involved in our project is food grade.
In addition, we pay special attention to treatment of experimental waste throughout the project:
The wasted petri dish are recycled in a harmless manner and placed in yellow garbage bags and placed under the "infectious waste" label after being sterilized using autoclave.
The waste liquid (no more than 85% of the volume) and empty reagent bottles are labeled with orange waste labels and placed under warning signs.
The waste liquid is mixed with disinfectant in a labeled bucket before being removed using specific sewage.
More detailed information is shown in the Lab safety section below.
Our current plan is only producing catechin as a final product using yeast.Our most ambitious goal ,which is not what we are doing now, is to add catechin into our bread during the fermentation stage using the modified yeast. We understand it will be an arduous and complicated process to devise a plan for a production line for “memory bread” and implement it in real life. To assess the feasibility we conducted a series of interviews and the results can be seen in the human practice part. ( https://2021.igem.org/Team:AISSU_Union/Integrated_HP）
In our research targeted to the public, we only collected data on attitudes of different age groups by questionnaires and interviews. Private information like names and address are not collected, ensuring the safety of the participants.In the interviews with experts, we also received professional instruction on safety precautions in fermentation as well as other stages of bread production.
According to our interview in the nursing home, we found the caretakers and the seniors preferred qualified medicine from hospitals. Therefore, we will try to cooperate with the local authorities and hospitals in the future to get the qualifications and permissions required before we sell the bread to the elders as a product.
The agentia and flavanols involved in our project are completely harmless and no strain or part involved exceeds risk group 1. As a result, all the experiments are conducted in S1 laboratory.
To conduct our experiment in a safe manner, all the team members went through training on the following topics:
-Wear lab clothes and gloves before experiments
-Operation safety for the apparatuses like clean bench, high speed refrigerated centrifuge and pipette and proper experimental techniques
-How we should behave in different areas of the lab to avoid contamination and leaking of microbes
In addition, apparatuses with potential danger are labeled with corresponding warning signs such as high temperature and high pressure and our team members are required to be supervised by a teacher when using the apparatuses.
What is mentioned above is only a small part of safety precautions in the laboratory and the translated complete regulations of our laboratory is shown in the PDF file below.
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Ando H, Lemire S, Pires D, et al. Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing[J]. Cell Systems, 2015, 1(3): 187.
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 Gibson D G , Benders G A , Andrewspfannkoch C , et al. and Cloning of a Mycoplasma genitalium Genome. 2008.