Overview
Our team’s contribution to the iGEM community focuses on advancing the understanding of how circadian rhythm disruptions, commonly caused by modern work and study pressures, can accelerate the progression of neurodegenerative diseases, specifically Alzheimer's disease (AD). By leveraging both experimental research and synthetic biology, we have developed the ROSE plan, which introduces an innovative approach to combat the adverse effects of circadian rhythm disruption.
Our contribution is multi-faceted, providing valuable insights and tools for future iGEM teams in the following ways:
1.In-depth Data on Rose Flavonoids and AD Progression: We conducted a comprehensive study using an AD mouse model with artificially disrupted circadian rhythms. Through these experiments, we collected significant data demonstrating the inhibitory effects of rose flavonoids on AD progression. This data can be utilized by other iGEM teams researching neurodegenerative diseases or those interested in the therapeutic potential of plant-based compounds.
2.Cell-Based AD Model and Troubleshooting Documentation: In our research, we recreated an in vitro AD cell model by intervening with neuronal cells and microglia. We meticulously documented the process, including challenges and troubleshooting steps, which future teams can refer to when developing similar models. This contribution of a reproducible cell model can facilitate research on neuroprotective agents and circadian rhythm-related disorders.
3.Identification of Key Genes and Metabolites: We have identified specific genes and metabolites through cell sequencing that are involved in AD progression under circadian rhythm disruptions. This genetic and metabolomic data is a valuable resource for future teams who are looking to explore molecular mechanisms or create targeted interventions for neurodegenerative diseases.
4.Functional Tea Product Development: Based on our findings, we are developing a functional tea drink infused with rose flavonoids aimed at alleviating the negative impacts of circadian rhythm disruptions. We are providing a detailed protocol for tea preparation, including optimal dosages and the scientific rationale behind the use of rose flavonoids. This could serve as a basis for other iGEM teams interested in creating biologically functional consumable products.
By sharing our research findings, detailed protocols, and experimental insights, we aim to foster further innovation in the field of neurodegenerative disease research and therapeutic interventions, particularly in relation to circadian rhythm disruptions. Our contribution is designed to provide valuable data and tools to fellow iGEMers, enabling them to build upon our work and develop new solutions for global health challenges.
Parts
Expression detection of the special functional protein and the related inflammatory pathway by western blot
The treated cells in Part 2 were collected and treated with lysis buffer, 200 μL of the 4× loadding buffer was added into each cell lysis sample, and we boiled and denatured the samples for 15 minutes. To quantify protein expressions of GRIN2B, associated proteins NF-κB, TNF-α and IL-1β, with respect to the control protein β-actin, WB is performed. 1× Tris-glycine electrophoresis buffer was filled into the electrophoresis unit, and samples and pre-stained rainbow protein marker were loaded onto the gel plates and electrophoresed initially at 120V for 15 min, then at 140V for about 30 min until the protein marker was completely separated. The PVDF membranes were prepared by one-minute immersion in anhydrous methanol. The membranes, sponges, and filter paper were submerged in a 1× transfer buffer, and the assembly sequences within the transfer apparatus was organized from the negative to positive pole as follows: sponge, filter paper, gel, membrane, filter paper, and sponge, and the membranes then underwent a 110V transfer for 1.5 hours. After transfer, the membranes were rinsed and incubated in 10% skimmed milk for an hour. The membranes were then washed using 1× TBST with 5 min for three times, which were further incubated with the primary antibodies for two hours. Later, the membranes were washed using 1× TBST buffer for five times, and next incubated with goat anti-rabbit IgG-HRP and 1× TBST for an hour. After three times of 1× TBST washing, 1 mL ECL developer for the membrane was prepared in darkness and added to the membrane,and chemiluminescence signals were captured with a designated imaging system, and the relative amounts of proteins in each group were quantified concerning β-actin expression.
