Safety is always an important consideration in any job and any product, and our project aims to provide new contact lens care solutions for keratitis treatment, so we have considered the multiple possible human effects of our product. This year, we analyzed the risks that could be encountered throughout the project and listed the following two items.
We worked in a BSL-2 laboratory in the Institute of Public Health. The laboratory is equipped with biological safety cabinet, emergency wind eye washing device, incubator, sterilization box, hand-washing pool, locker, storage cabinet, lighting equipment, special work clothes, gloves, shoe covers and other laboratory equipment.
The laboratory is equipped with autoclaving equipment and we strictly followed the BSL-2 laboratory protection requirements and safety rules. Before starting laboratory work, all our team members received comprehensive laboratory training, including laboratory skills, safety training, and proper use of laboratory equipment. In order to avoid unexpected damages, appropriate safety measures such as wearing gloves, goggles, and laboratory coats, were applied in experiments to ensure protection from biotic and abiotic hazards.
In the early stages of the experiment, we insisted on working in groups of three and supervising each other to ensure that the experiment be carried out safely. The teachers checked the safety of the laboratory and regulated the behavior of the laboratory team members on a regular basis. Before entering the laboratory, everyone must stick to the laboratory rules. All laboratories are equipped with complete safety emergency equipment and have prominent safety signs on large instruments. When entering the cell laboratory, we had to wear extra shoe covers and head coverings, and the cell laboratory was regularly UV-sterilized to ensure no contamination.
At the same time, we paid special attention to the safety of experiments related to cell biology, and conducted experimental operations in ultra-clean platforms to reduce the possibility of contact with viruses and cells, prevent contamination and ensure safety. Our experienced instructors provided us with guidance and specifications for experimental operation and safety management to ensure smooth experimental processes for team members in a safer experimental environment. To ensure that our team members work in a safer laboratory environment, we strictly follow the safety and security rules of the iGEM competition. The reagents and drugs were classified, and dangerous reagents were placed in dedicated storage cabinets with warnings. All of our experiments this year were low risk basic biology experiments using the organism Escherichia coli. For the use of low-risk organisms, we conduct most experiments in biosafety cabinets to avoid the generation of aerosols. For products containing genetically engineered organisms, specific biosafeguards are implemented to limit their spread. Flammable materials are stored separately on shelves that have undergone laboratory safety checks. Infectious health care waste is placed in specific bins and processed by approved disposal plants.
Laboratory safety is inseparable from the efforts of each team member, and compliance with laboratory rules is necessary to ensure the smooth progress of the experiment.
Due to the need to ensure the safety of the process of controlling protein production by E.coli during the production process, we designed a growth switch to control bacterial growth at different stages of the product life cycle. The toxin antitoxin system HicA-HicB was used to control bacterial growth. HicA is flanked by FRT sites that can later be deleted by FLP recombinase. When the HicA toxin and HicB antitoxin were combined, the bacteria remained dormant, while the FLP gene regulated by the CI857 thermosensitive promoter knocked out the HicA toxin gene and returned to normal growth. This design allows us to control the production and storage of bacteria and avoid contamination.
To prevent uncontrolled expansion of our engineered bacteria, we designed the population sensing system.
The mechanism of population sensing is achieved through the regulation of AHL signaling molecules. In our design, when the concentration of AHL reaches a certain threshold, it crosses the membrane into the bacterial body, activates the expression of downstream genes after binding to LuxR, and produces in vitro E-cleavage protein. When the protein reaches the threshold concentration, the engineered Escherichia coli is lysed and killed, so that the population density of Escherichia coli is maintained at a certain level.
This precise regulatory mechanism helps to prevent unexpected transcription and expression activities and prevent excessive proliferation of Escherichia coli, thus ensuring the safety of the experiment.
In the process of this year's project, the DMBT1 protein produced by the engineering bacteria we used is a human protein, which is widely present in the human body and does not produce immune response. At the same time, in order to simulate the antibacterial effect of the product in the human eye, we used human corneal epithelial cells to conduct experiments, so as to make the application of the product more secure.
To ensure that our contact lens care solution did not cause any harm, we chose to extract DMBT1 protein from the exosomes of bacteria and add it to the care solution to prevent the bacteria from contacting the eye. Our nursing solution cannot be purchased directly from pharmacies, and patients who use this product at the same time must consult their doctor regularly to ensure the efficacy and safety of the nursing solution.