In our 2024 iGEM project, we have created an active drug screening platform in HEK293T cells that utilizes the MT-CRE pathway. This platform is able to respond significantly to the anti-insomnia properties of various essential oils (or aromatherapy). In order to better improve the sensitivity and discriminability of the platform response, we started from three known pathways, cAMP,ERK and Ca²⁺, respectively, and designed regulatory binding sites with different multiplicities. Finally, CRE (cAMP response element) was determined to be responsible for the response of this project.

As a therapy project, we have prioritized the safety issues as follows:
1. E. coli strains (DH5-alpha and Stbl3) were utilized for standard molecular cloning procedures. The strains employed in our research have not been reported to cause disease in immunocompetent adult humans, thereby presenting a minimal potential hazard to laboratory personnel and the environment.

2. HEK293T cells were utilized to demonstrate the feasibility and efficiency of the newly constructed plasmid. The 293T cell line is derived from 293 cells and incorporates the E1A gene from adenovirus. Although adenoviruses can cause diseases in humans, HEK293T cells are considered entirely safe for laboratory use. Furthermore, these cells cannot survive outside of a controlled laboratory environment.

3. All genes cloned into pcDNA3.1 and pRK300 are non-conjugative, thereby preventing the horizontal transfer of our genetic components. We employ the P_min and 4×CRE as promoter, which ensures that our bacterial system will not produce significant amounts of gene products unless expression is induced.

4. All components utilized in our processes are assured to be safe for human use and will not contribute to any biological pollution in the environment.

1. All our operations comply with the safety regulations of the laboratory safety.

2. We adhere strictly to the guidelines outlined in the World Health Organization's Laboratory Biosafety Manual, in conjunction with the explicit instructions provided by our instructors. It is mandatory for us to have a comprehensive understanding of the experimental principles and methodologies prior to embarking on any operational procedures. This approach ensures adherence to the highest standards of biosafety and promotes accurate and efficient experimental execution.

3. All members of our experimental team have undergone comprehensive laboratory security and safety training, ensuring a profound understanding of fundamental safety protocols, pertinent policies, and regulations. This rigorous training program equips us with the knowledge and skills necessary to mitigate potential hazards and maintain a safe and secure working environment.

4. Completion of a passing examination is mandatory before individuals are granted permission to operate in a Biosafety Level 1 (BSL-1) laboratory. Additionally, a specialized training program has been designed to prepare individuals for access to the BSL-2 laboratory, where our mammalian cell cultures are maintained, ensuring adherence to the highest safety standards.

5. Prior to utilizing a new machine, we thoroughly review the instructions multiple times to ensure a comprehensive understanding of the proper operating procedures. Furthermore, we complete our initial operation under the direct supervision of our safety managers, ensuring we are fully competent before independently manipulating the machine.

6. All waste materials undergo rigorous sterilization processes and proper treatment measures before being dispatched to qualified disposal companies. Specifically, abandoned bacterial cultures are subjected to high-temperature and high-pressure sterilization protocols to ensure complete inactivation, prior to their disposal. This specialized approach guarantees the safe and environmentally responsible handling of all waste generated during our operations.

7. 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.