Original parts
Minimum TATA-box promoter.The minimum TATA promoter is a eukaryotic DNA sequence that indicates a transcription start site. The minimum TATA promoter shows low levels of transcription at baseline. (Part:BBa_M50098)
Our improvement
Figure 1.The structure of our improved part
Our improved part is 9XGlucose Sensing Promoter(BBa_K3064012). Since there is high blood glucose concentration in diabetics, we hope that our promoter can react to high blood glucose.T his year we have made a series of modification based on the Minimum TATA-box promoter designed by Daniel Tang of Stanford BIOE44 - S11.(BBa_M50098). In order to realize the glucose-sensing function, we added glucose-sensing fragment, ChREBP. Transcription factor -- ChREBP -- can be effectively expressed with high blood glucose. Meanwhile, heterodimer which consists of ChREBP and Mlx can combine with gene promoter CHoRE to induce gene transcription. Therefore, we choose CHoRE as the promoter of engineered plasmid. In addition, we increase the number of CHoRE to make sure that engineered gene will be activated by particular high blood glucose concentration instead of normal blood glucose concentration.
Besides, Due to the low efficiency of TATA box promoter, we shorten the sequence into only minip. The function of minip is almost similar to TATA’s. As a result, minip can be considered as a smaller version of TATA box promoter, with shorter length and almost the same function.
In conclusion, we connect 9 CHoREs with minip to indicate a transcription start site. With this design, we plan to realize the promoter’s glucose-sensing function and enhance its initiation strength and thus make sure our engineered protein predator play its role when there is high glucose concentration.
We believe that our improvement promote a glucose-sensing promoter with high efficiency of expression and high sensitivity to blood glucose concentration. With this promoter, our protein predator can better play its role and fight against T2D.
Experiment methods
First we choose luciferase as the reporting system.We separately transfected plasmids PGL3-9XGSP and PGL3-minP into HepG2 cells. After culturing these cells with glucose-free culture, we created low glucose concentration environment for cells and then stimulated cells with 20 mM glucose concentration culture. After 48 hours, these cells were gathered and dissoved for luciferase expression test with Dual Luciferase Reporter Gene Assay Kit of Beyotime company.
Figure 2.Transfection and Test with luciferase
In other experiment, we adopt GFP to report the expression of the improved promoter. At the very first beginning, we starved the HepG2 cells with DMEM for 2 hours before transfection begins.After transfection 12h, we starved the cells with glucose-free culture and stimulate with 20mM glucose concentration culture after 6 more hours. Glucose stimulation intensity was controlled at 20mM. Samples were tested after transfection at different times.But this time we choose GFP/mcherry as indicators to show the expression level and use photograph to show the tendency of indicators.
Figure 3.Transfection and Test with GFP
Data
The experiment data and results are shown on the part registry. Parts:9xGSP.