This year, we simultaneously utilized some of the basic elements, namely our previous designs, and also some new innovative designs to finish our project. Compared to the components that simply leads to an increase in the expression of insulin in previous years, we have added a braking system to the design in order to achieve more precise control of insulin concentration.

Fig 1. Composite parts

Our contribution is the construction of the above composite parts, which are composed of multiple basic parts to form plasmids that perform certain functions. All of these may be helpful to other teams, and we hope it will make contributions to the iGEM community.

 (For detailed information, please visit the PARTS in our wiki.)

pcDNA3.1-LOV-VP16 is a specially designed expression vector, integrating the superior performance of pcDNA3.1 plasmid with the unique function of LOV-VP16 fusion protein. pcDNA3.1, a widely-used high-efficiency expression vector in mammalian cells, harbors a potent CMV promoter, driving high expression of foreign genes in various mammalian cell lines. The LOV domain, derived from a photosensitive protein, senses blue light signals and undergoes conformational changes; the VP16 domain significantly enhances gene transcription. When fused, the LOV-VP16 protein enables precise control of target gene expression under blue light regulation.

Fig 2. Structure of the LOV-VP16 plasmid

GIPpromoter-GI-GAL4 plasmid is a specialized vector integrating a specific promoter, reporter gene, and transcription factor. It employs the GIP (Glucose-dependent Insulinotropic Polypeptide) promoter, a DNA sequence with tissue specificity and regulatory properties, capable of initiating downstream gene expression in response to glucose concentration changes. In this plasmid, the GAL4 domain is linked to or regulated by the reporter gene, enabling it to drive reporter gene expression when GAL4 is activated, such as through binding to specific ligands or interacting with other transcription factors.

Fig 3. Structure of the GIPpromoter-GI-GAL4 plasmid

The GIPpromoter-LOV-VP16 plasmid integrates the LOV domain, allowing precise gene expression regulation via blue light. VP16, a powerful transcriptional activator, forms a LOV-VP16 fusion. Blue light triggers a conformational change in LOV, potentially activating VP16 and driving downstream gene expression.

Fig 4. Structure of the GIPpromoter-LOV-VP16 plasmid

In the GIPpromoter-sponge plasmid, the sponge sequence is designed to express a piece of RNA that specifically binds to a large number of specific microRNA molecules, thereby inhibiting miRNA activity. By integrating the sponge sequence into the plasmid, the level of specific RNAs within the cell can be regulated, which in turn affects the expression of related genes.

Fig 5. Structure of the GIPpromoter-sponge plasmid

The 5XUAS-insulin-miR-2BS plasmid is a specialized vector that combines specific enhancer sequences, the target gene, and microRNA binding sites. Here, the transcriptional activation efficiency was enhanced by tandemly linking 5 UAS sequences, making the expression of downstream genes more sensitive and efficient. Additionally, we have also integrated miR-2BS after the luciferase reporter gene, allowing miRNAs to bind to it and to regulate the expression of the luciferase reporter gene.

The 5XUAS-insulin-miR-2BS plasmid is a specialized vector that combines specific enhancer sequences, the target gene, and microRNA binding sites. Here, the transcriptional activation efficiency was enhanced by tandemly linking 5 UAS sequences, making the expression of downstream genes more sensitive and efficient. Additionally, we have also integrated miR-2BS after the luciferase reporter gene, allowing miRNAs to bind to it and to regulate the expression of the luciferase reporter gene.

Fig 6.Structure of the 5XUAS-LUC-miR-2BS plasmid