UAS (BBa_K758003) is a piece of small DNA sequence that can be recognized and combined by GAL4 DBD. Team iGEM12_KIT-Kyoto repeated UAS 5 times when using it. In our design, considering of that the mutual effect of our blue light protein would reduce the efficiency and the whole pathway is relatively long, while the react time of our system should be as short as possible, we have made some alterations.
Firstly, we have repeated the UAS before promoter for 9 times. The purpose of constructing 9XUAS-Ad-LUC (BBa_K3734032) and 9XUAS-Ad-insulin(BBa_K3734033) is to enhance the recognition and combination of GAL4 DBD, improve the efficiency of downstream target gene activation.
Secondly, we have used Ad promoter as promoter to make it able to function in eukaryotic cells and improve the expression efficiency after the activation of VP16.
Fig.1 The model diagram of 9XUAS-Insulin of 9XUAS-Insulin
Fig2.The expression level of inducible promoter CHREBP was respectively analyzed at 72h in 25mM, 5.6mm and 0mM glucose cultur.
Fig3.Changes of LUC expression over time after blue light irradiation with different glucose concentrations
Firstly, we used report gene LUC as the bottom target gene, positive and negative control groups were set up. The result suggested that the e fficiency of 9XUAS part has a big improvement. It can meet the demand of shortening the reaction time.
Fig2.Light controlled system testing experiment
Then, we changed the bottom target gene into insulin gene and proved that the insulin can express successfully and process maturely then secrete outside the cell. The efficiency of 9XUAS is high enough.
Figure 3: Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively
Conclusion: We enhanced the method and the supporting components of UAS, repeated UAS sequence for 9 times and changed the promoter after UAS into Ad promot er. We improved the efficiency of the recognition and combination of GAL4 DBD and UAS along with the efficiency of activating the expression of downstream gene s to meet the requirement for this loop to be put into use. Meanwhile, we have provided a more efficient tool for afterwards iGEM teams .
In the designing stage of our project, we decided to have a NOT part. It is able to: (1) Inhibit the insulin expression in a feedback system to form a close loop, makes it more automatically; (2) Stop insulin from secreting more to prevent hypoglycemia, makes it safer.
We chose protac in the first place. It can connect target protein and E3 enzyme to pantizate the target protein and degrade it. However, as the design went on, we have discovered that protac are mostly small molecules, it is hard to express directly by cells and needs exogenous factors, which is contrary to our automatic and convenient idea.
Fig.4 The model diagram of protac
Then we discussed degron, it also acts on target protein and makes the target protein able to be degraded, but we wanted a root method for inhibiting the expression of insulin.
In the end, we used NO.21 miRNA (miR21), it can inhibit the target gene expression and speed up the degradation of mRNA
Fig.5 The model diagram of pG-super-miR21
Fig.6 The model diagram of TRE-mCherry-miR21
When designing the experiment, we detected LUC/REN value 48 hours after the transfection. miR21 has suppressed 40% of expression, which is not ideal for our design.
Fig.7 miR21 inhibited Luc expression 48h after transfection
Considering it might be because of the accumulation of LUC after expression, we did a 24-hour group. It turned out that miR21 has suppressed 90% of expression, which fits our design perfectly.
Fig.8 miR21 inhibited Luc expression 24h after transfection
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