We worked exclusively with Biosafety Level 1 organisms, including Escherichia coli Nissle 1917, Escherichia coli DH5-α, and Lactobacillus plantarum L168. In addition, the Parts in our project were all Biosafety Level 1 Parts as well.

It has been demonstrated that NAD+ improves mitochondrial activity. In system one, we chose Lactobacillus plantarum L168, a common probiotic, as the chassis, and PLDHLH673 as the vector. The engineered bacteria boost NAD+ expression by overexpressing the pncA gene.

For biosafety, we designed a suicide switch that can stop them from overgrowing inside the human body and prevent their release into the environment. The suicide switch is activated by a transferable nisin-controlled expression (NICE) system based on the combination of the nisA promoter and nisRK regulatory gene.

Ingested heavy metals frequently target mitochondria. In System two, we chose Escherichia coli Nissle 1917, a common probiotic, as the chassis, and PET28a as the vector. The engineered bacteria sense the heavy metals and produce metallothionein (MT) to adsorb them.

Similar to the first system, we also designed a suicide switch in System two to stop them from overgrowing inside the human body and prevent their release into the environment. The suicide switch is activated by arabinose based on the BAD promoter and MazF gene.

Elevated lactate levels are the most common marker of mitochondrial dysfunction. In system four, we chose Escherichia coli DH5-α, a common probiotic, as the chassis, and pSB4K5 as the vector. The engineered bacteria can sense the variation of L-lactate levels, express the galactosidase, and react with the X-Gal on the testing strip.

Since the plasmid we designed is too large to add the sequence of the suicide switch, our current design does not include one for the engineered bacteria on the testing strip. The whole team has been thinking about approaches for the environmental release all the time and has gone through solutions, from adding a small kill switch to using auxotrophic strains that can only grow on the testing strip. However, none of them turned out as we assumed, and we are still trying to look for a suitable solution.

Currently, our solution is a Lactate Test Kit that includes a safety cell to prevent the engineered bacteria from entering the environment. The recess of the cell contains disinfectants, and users are instructed to insert the testing strip into the recess after the test. We also plan to provide future users with a recycling pouch and instruct them to place the testing kit into it for further processing in the pharmacy or hospital where the product was purchased.

Although we have not got a perfect solution, it should be noted that our engineered bacteria are currently only grown in the laboratory and will not be released into the environment. We will ensure that our bacteria are used in the laboratory only before we find the best solution for this problem, and we firmly believe we will work it out.

Under the guidance of our PI, Professor Xingyin Liu, an expert in the department of pathogenic microbiology at Nanjing Medical University, we have fully considered possible risks and developed plans and solutions at the design stage. During the experiment, we can go to our laboratory technician Xiaoting Luan and the 2021 NJMU-China team member Qinwen Wang for help. Xiaoting Luan and Qinwen Wang are lab members of Xingyin Liu Lab and worked as advisors for our project this year.

In addition, our school, Nanjing Medical University, has a consummate laboratory management system , and we can always turn to the school for help if the risk presented turns out to be beyond our supervisors.

Each of our wet lab members has passed the school's Experiment Safety Assessment and got certification one to two years before the iGEM competition. Before conducting experiments to test our design, our vice leader, Xinyuan Wang, who has two years of experience in the laboratory, hosted a lecture on laboratory safety. The activity allowed us to double-check the safety of our project based on a relatively comprehensive set of standards. Besides, we all received the instruction and training of our PI before we carried out any experiment. In order to use the lab during the summer break (July and August), we filled out the application form and got the consent of the college's laboratory management department.

the lab safety certification

the lab safety lecture

During the experiment, we have been following the rules below:

A team member is performing the experiment safely

We made a safety handbook which discusses the hydropower safety, chemical safety, fire safety, and biosafety in our laboratory with the aim of emphasizing lab safety and also acting as a reference to future teams.

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In order to carry out our human practices safely during the Covid-19 pandemic, we participated in activities that required cross-province traveling virtually throughout the competition. Meanwhile, we maximized safe commutes by reminding every team member who was attending an offline meetup or visiting the institution for autistic children of the traffic rules.

Human subjects research safety

Since we wanted to build a project that is responsible for the world, we made sure that we followed regulations on social science ethics in all of our human practice activities involving a human subject. The rules were discussed thoroughly in our weekly meetings within the team, and team members took turns presenting related laws and regulations each week. Besides the regulations in our country and the institutions we visited, we also looked at the Code of conduct for Social Science Research of UNESCO, which helped us double-check the ethicality of our conduct.

Additionally, in every one of our four interviews, we put great importance on informed consent. To achieve that, we handed out related forms to the subjects and collected the documents afterward to see if they were willing to participate in our activities. We also provided detailed instructions and explanations of our activities to our subjects so they could better understand beforehand. Meanwhile, the photos we presented in our wiki are all used with consent and permission. Pictures of children are used with permission from both the children and their parents.

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Our questionnaires were also designed to be anonymous to protect privacy as well, and the exact answer of the participants are now revealed in our analysis. We tried to include more objective questions instead of subjective questions to reduce potential insecurity among participants of exposed privacy. All of the data we collected was well-protected and we assured our participants of that before carrying out our activities.