The aim of our project is to construct a designer cell that expresses insulin in response to high glucose level. The expression of insulin is controlled by an AND circuit, a glucose-dependent promoter and a glucose-dependent switch for blue light. In addition, we also designed a NOT circuit to prevent the excessive insulin expression and subsequent hypoglycemia when glucose levels drop. The NOT circuit includes a miRNA-binding element at the 3’-UTR of insulin coding region, miRNA and sponge. To easily monitor the precise expression of insulin, we used luciferase reporter in the experiments. The expression plasmids for insulin and luciferase were shown Figure 1.
Figure 1. The expression plasmids for insulin (A) and luciferase (B)
In this project, we chose GIP promoters to control the expression of galactose-responsive transcription
factor GAL4 [Saccharomyces cerevisiae S288C], VP16 [Human alphaherpesvirus 2], and sponge, as it was
reported that GIP promoter can be activated by glucose.
To confirm that the GIP promoter can be activated by glucose, we constructed a plasmid (2401), in which
the expression of luciferase is under the control of GIP promoter. We then transfected 2401
pGIP-luciferase plasmid into 293T cells cultured under different glucose concentration (0, 1, 5, and 25
mM), and the enzymatic activity of luciferase was measured. As shown in Figure 2, the luciferase
activity increased in response to higher glucose levels.
Figure 2. Glucose activated the GIP promoter in a dose-dependent manner
As shown in Figure 1, the expression of insulin/luciferase was controlled by a 5XUAS promoter, which can be activated by GAL4-VP16 transcription factor. To confirm the activation of 5XUAS promoter by GAL-VP16, we transfected 5XUAS-Luciferase (2409) in the absence or presence of plasmid expressing GAL4-VP16 (2404) into 293T cells, and the enzymatic activity of luciferase was measured. As shown in Figure 3, there is about 2000 fold increase of luciferase activity after expression of GAL4-VP16. Therefore, our data verified the activation of 5XUAS promoter by the GAL4-VP16 transcription factor.
Figure 3. Activation of 5XUAS promoter by the GAL4-VP16 transcription factor
In our design, the DNA-binding domain (Gal4) and transactivation domain (VP16) of transcription factor Gal4-VP16 were separated and fused to GI and LOV, respectively. It is proposed that the GI-Gal4 (2406) and LOV-VP16 (2407) will form an active transcription factor in the presence of blue light, to activate the expression of proteins under the control of 5XUAS. To test this, we co-transfected 5XUAS-Luciferase and plasmids expressing GI-Gal4 and LOV-VP16 into 293T cells, and cells were cultured in the absence or presence of blue light. As shown in Figure 4, there is about 300-fold increase in luciferase activity when the cells were exposed to blue light for 15 minutes. Our data thus verified that blue light can stimulate the formation of an active transcription factor from GI-Gal4 and LOV-VP16.
Figure 4. Blue light can stimulate the formation of an active transcription factor from GI-Gal4 and LOV-VP16
As GIP promoter responses to an wide range of glucose levels, we sought to design a switch in response to a specific glucose concentration. To this end, we have collaborated with Luzai Technology Company to construct a blue light switch under the control of glucose concentration. As shown in Figure 5, a digital glucometer is modified to a switch, when the glucose concentration reached certain level (in this case, it is 10mmol/L), the switch was turned on and the blue light was subsequently turned on, which stimulate the formation of an active transcription factor from GI-Gal4 and LOV-VP16 (Figure 5).
Figure5. Blue light strument under the control of glucose concentration
The small box below the picture is a glucose detection instrument. When a test strip is inserted and immersed in a glucose solution, it can detect the concentration of the glucose solution and display it on the monitor screen on the left side of the picture. On the right side of the picture is a blue light instrument that can control the blue light switch based on glucose concentration.
In the above AND circuit, we demonstrated the feasibility of insulin expression when glucose concentration increases. However, using the previous circuit alone may lead to excessive insulin expression and the risk of hypoglycemia, as it takes for hours for Gal4-VP16 to degrade. To solve this problem, we designed a miRNA system to inhibit the expression of insulin/luciferase. In this system, the miRNA-binding site was placed within the 3’-UTR of insulin/luciferase. As shown in Figure 6, co-transfection of a miRNA-expressing plasmid significantly inhibited the expression of luciferase induced by Gal4-VP16.
Figure6. Inhibition of luciferase expression by miRNA
In order to prevent miRNA from exerting inhibitory effects on insulin/luciferase expression, we introduced sponge, which can bind miRNA and disinhibit the miRNA effect. As shown in Figure 7, co-transfection of sponge attenuated the inhibitory of miRNA on the expression of luciferase.
Figure 7. The disinhibition effect of sponge
Ideally, the sponge functions when the glucose level is high and is dysfunctional when the glucose level drops. To this end, we placed the sponge under the control of GIP promoter. As shown in Figure 8, the disinhibitory effects of the sponge is significantly higher in 25 mM glucose than 1mM glucose.Therefore, The sponge RNA exerts its function in a glucose-dependent manner.
Figure 8. The sponge exerts its function in a glucose-dependent manner
In summary, we have validated the AND circuit and NOT circuit in 293T cells. Both circuits function as expected. We are thus able to synthesize insulin when the glucose level reaches a specific level by using blue light together with a GIP promoter. We are also able to stop insulin expression when glucose level drops by using a miRNA-sponge system.