The uncoupling protein UCP1, the most well-studied mitochondrial uncoupler that regulates thermogenesis and cellular energy expenditure(Kozak & Anunciado-Koza, 2008), has been used by the team iGEM08_Valencia in their project to control the temperature of the yeast culture. In their documentation of BBa_K141000, they mainly provided functional data on how the overexpression of UCP1 would affect the temperature and the growth kinetics of yeast. Building on this, we supplemented some useful data regarding where UCP1 is located and how it affects cell energy expenditure in HEK-293T cells, a commonly used mammalian cell line.


Methods and Results

Methods
To visualize the subcellular location of UCP1, we fused an EGFP protein on the N-Terminus of UCP1 (EGFP-UCP1). HEK-293T cells were transfected with this EGFP-UCP1 expressing plasmid for 48 h before the localization of EGFP-UCP1 was observed via widefield fluorescent microscopy and live-cell confocal imaging. We also analyzed the glucose consumption of the transfected cells by measuring the glucose levels in the culture medium. This analysis represented the level of cellular energy expenditure.

Results
Both wide-field fluorescent imaging (Figure 1a) and live-cell confocal imaging (Figure 1b) showed highly specific colocalization of EGFP-UCP1 signal with mitochondria markers (MTS-mcherry, Figure 1b). Moreover, cells transfected with pNC088 (P_CMV-Pdp1NTD-EGFP-UCP1) showed a significantly higher glucose consumption compared to the control cells transfected with pcDNA3.1(+) vector (Figure 1c), suggesting a significantly improved energy consumption in these cells.

We are the first team to introduce the protein-delivering Photorhabdus Virulence Cassette (PVC) system into the iGEM community. This system was recently reported and engineered by Kreitz et al. (Kreitz et al., 2023) as a new protein delivery approach to directly inject proteins into mammalian cells in a target-specific manner. Our team created and documented different functional units of PVC in the Parts Registry and created multiple parts to help future iGEM teams engineer the PVC coat and PVC payload. We also included a Golden Gate-based pvc13 construct, that may possibly simplify the cloning process for PVC tail fiber engineering. Additionally, we created a part collection containing all the necessary components to assemble PVCs and the PVCs we used in our project.

The protocol that we used to construct these parts is documented on the Experiments page, the parts can be found on the Parts pages (Parts Overview, Basic Parts, Composite Parts, Part Collection).

Our goal is to create a PVC particle that targets adipocytes using UCP1. However, this has presented numerous challenges during the purification process. As a new field, there are limited references to using PVCs as a programmable protein delivery tool, which has made the process even more difficult. Despite these challenges, we have successfully engineered the PVCs and documented every step of the procedure, including plasmid construction techniques and temperature control. We have also included unsuccessful steps and techniques, along with tips and warnings, on our Experiments pages. This documentation will be useful for future iGEM teams looking to replicate our work.

To provide a more direct visualization so others can understand PVCs better, we have created an open-source 3D model using Solid Works software. This model can be used by others to create a miniature PVC replica quickly using a 3D printer. This will help users gain a better understanding of the structure and physical workings of PVCs.

We have created a card game called ARROW Forward to promote synthetic biology. The game is inspired by the popular game Saboteur and engages players in a thrilling research adventure. Players assume the roles of researchers and strive to reach their respective research destinations represented by three mysterious basic cards. Throughout the game, players collect objective, link, and action cards, which enable them to advance their research by acquiring essential components, brainstorming ideas, optimizing projects, and conducting experiments.

ARROW Forward aims to demonstrate the challenges and significance of synthetic biology. By playing this interactive game, players can gain a deeper understanding of the iGEM and synthetic biology projects, as well as the collaborative nature of scientific research. We hope that through this innovative approach to education, more individuals will be inspired to explore the world of synthetic biology and contribute to its advancement. See our Education page for more information about this game.

Our team has created a curriculum framework on synthetic biology specifically designed for high school students. Our goal is to provide students with a step-by-step learning experience that will enable them to develop a deeper understanding of the subject, spark their interest in the field, and inspire them to potentially pursue a career in synthetic biology research. Our hope is that this curriculum framework will contribute to the advancement of synthetic biology.