Difference between revisions of "Part:BBa K3734033"

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

We used human mature insulin ELISA kits to detect the level of insulin secreted by engineering cells.

We use the gene of the downstream target with the insulin gene, and successfully proved that insulin can be successfully expressed, processed and secreted out of cells, and 9XUAS is efficient enough.

Fig.2 Insulin and absorbance standard curve

Fig.3 Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively

Because we repeat UAS nine times, we should pay attention to avoiding duplicate fragments when designing primer. If you can't avoid duplicate clips, please note the times that UAS in PCR products has been repeated.Insulin is not suitable for long time preservation under the best culture temperature of cells (37℃). In the mean time, cells would also consume some of the expressed insulin. So the best tst time for Insulin is approximately 24 hours after transfection, it is relatively shorter than the test interval of LUC.

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[2]Mingqi Xie, Haifeng Ye, Hui Wang.β-cell-mimetic designer cells provide closed-loop glycemic control[J].Science.2016 Dec 9;354(6317):1296-1301.