Our approach involves the creation of a genetic construct that incorporates the EFS promoter, uORF, Reg3g CDS, YFP, and a terminator. Our team includes multiple parts from the iGEM distribution kit to achieve our final result. The Reg3g CDS, crucial for its therapeutic potential, is produced via PCR. To assemble these elements, we employed Golden Gate cloning with Type II S enzymes, which enables sequential ligation of different sequences. Initially, we constructed level 0 (L0) structures for each part, with each L0 part containing a single DNA fragment, such as the EFS promoter. The L0 plasmids from this year's distribution kit featured two BbsI sites, which we utilized to create overhang sequences facilitating the assembly into level 1 (L1) transcriptional units. BsaI was employed to cut the L0 structure, leaving behind sticky ends composed of 4 base pair long single strain. Sticky ends are also known as "overhang" , the front overhang of a part is complementary to the tail overhang of the former DNA, which enables the connection of EFS to uORF, uORF to Reg3g, and so forth. Overexpressing the Reg3-ʏ gene holds excellent potential for T1D patients, as it promotes beta cell regeneration and modulates immune responses. This overexpression activates the “Janus kinase 2/signal transducer” and “activator of the transcription 3/nuclear factor κB signaling pathway”, enhancing beta cell regeneration while attenuating lymphocyte infiltration. Research by Xia et al. (2016) highlighted the role of Reg3g in boosting β-cell regeneration. Reg3g expression reduces lymphocytic infiltration, indicating reduced autoimmune activity. It achieves this by inducing regulatory T cells and tolerogenic immature dendritic cells, thereby dampening autoimmunity. Additionally, Reg3g expression promotes beta cell regeneration through the Janus kinase (JAK) pathway, a vital signaling system in this process. To fine-tune the overexpression of Reg3g, we manipulated Upstream Open Reading Frame (uORF) sequences. Our study explores two transcriptional units—one with a single uORF sequence and another with four. By comparing their efficiency, we aim to identify the optimal transcriptional unit for safeguarding T1D patients. In our pursuit of beta cell protection, another avenue we explored involves RNLS (renalase). Deleting RNLS has the potential to protect beta cells and alleviate T1D by mitigating endoplasmic reticulum (ER) stress and inflammation-induced cell death. We adopted an approach to overexpress mutated RNLS, creating transcriptional units with a constitutive EFS promoter, a 5'UTR to enhance translation efficiency, and RNLS binding to yellow fluorescent protein (YFP) for easy monitoring of gene expression. Furthermore, we delved into the structure and function of renalase, a protein encoded by RNLS. Renalase is an enzyme that catalyzes the oxidation of specific amines, utilizing NADH as a cofactor. It contains two essential domains: a substrate-binding domain and an amino oxidase domain, running from the 102nd to the 297th amino acid. This amino oxidase domain serves a dual role, binding necessary molecules like FAD and NAD and catalyzing specific amine oxidation. By combining these genetic approaches and insights into Reg3g and RNLS, we aim to advance the field of beta cell protection and T1D management.

As beta cells are necessary for maintaining insulin production in T1D patients, we overexpress the Reg3-ʏ gene. It could regenerate beta cells by facilitating leptin secretion.