For our 2022 iGEM project, we developed a synthetic biomedical tattoo using engineered cells that would monitor the rising glucocorticoid level induced by chronic stress and respond accordingly with a visible output. For this purpose, we engineered a novel synthetic transcriptional factor that could translocate from the cytosol into the nucleus upon glucocorticoid stimulation and initiate reporter gene transcription. We confirmed that the cells carrying this synthetic transcriptional factor and its corresponding reporter cassette could respond dose-dependently to glucocorticoid stimulation. We also showed that the transgenic tdTomato or tyrosinase expression under the control of such a glucocorticoid-responsive circuit could generate either fluorescent or colorimetric signals detectable by the naked eye. The response and production would be achieved by different modules, including LBD of NR3C1, tetR/tetO, tdTomato, tyrosinase, etc. Putting safety first, we considered the safety issues as follows:
1. E.coli was used to perform molecular cloning. The stbl3 strain we use has not been reported to cause disease in immunocompetent adult humans consistently, and it does present a minimal potential hazard to laboratory personnel and the environment.
2. HEK293T cells were used to demonstrate the feasibility and efficiency of the newly constructed plasmid. 293T cells derive from 293cells and are embedded with gene E1A of adenovirus. Though adenovirus could lead to diseases people, the 293T cells are entirely safe. And they are unable to survive when they escape from the lab.
3. All genes cloned into pcDNA3.1 are non-conjugative, preventing the horizontal transfer of our parts. We use CMV as promoter, which guarantees that our bacteria won’t produce large amounts of gene products if we do not induce the expression.
4. All the parts we use are guaranteed to be safe for humans and won’t cause any biological pollution to the environment.
5. We plan to use microencapsulation technology to embed engineered cells to prevent cells from leaking from the injection site and causing harm to the human body.
Although E.coli stbl3 strains might present a minimal potential hazard to laboratory personnel and the environment by spreading resistant genes to the public, it does present a minimal potential hazard to laboratory personnel and the environment. Since most of these cells will be transformed with plasmids with resistant selection markers, if the bacterial wastes are not properly treated, they may spread the resistance genes into the environment. So limits were set to avoid this kind of pollution.
HEK-293T cells are unable to survive when they escape from the lab. However, accidentally releasing these cells into the bloodstream might cause harmful health issues. So members were careful when modifying cells.
Meanwhile, the cells we use still have the potential to divide. The ethical review of cells with the ability to divide is more stringent. We should restrict the division of engineered cells to guarantee the safety. Another factor is that we should ensure compatibility with other operations like blood draws and surgeries. More lab works need to be done to reduce risks.
1. All our operations comply with the safety regulations of the laboratory safety manual.
2. We strictly follow the guideline of the WHO lab biosafety manual and the instruction of our instructors. We are required to understand the experimental principle and method completely before the operation.
3. All our experiment members have received comprehensive lab security and safety training to ensure that we are well aware of basic safety protocols and related policies and regulations.
4. Exam should be passed before getting permission to operate in the BSL-1 lab alone. Additional training is planned specifically to obtain access to the BSL-2 lab, where we culture our mammalian cells.
5. Before we use a new machine, we will read repeated instructions to ensure we grasp proper actions. We must finish our first operation under the direction of our safety managers before we manipulate the machine by ourselves.
6. To minimize the damage caused to us by the experiment, we used a blue imager to replace the UV imager and the UV glue cutter. Also, we used non-polluting nucleic acid dyes when preparing agarose gel and agarose gel electrophoresis.
7. All wastes are sterilized with proper treatment and finally sent to qualified companies. The abandoned bacteria can only be thrown away after high temperature and high-pressure sterilization.
8. Members from our Institutional Biosafety Committee will review our research plan and support our risk management. And our laboratory safety management team is experienced and sound. We will seek our laboratory safety manager for guidance when we discover a hazard or risk in our project.