Revision as of 20:01, 21 October 2019

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. Sequence and Features

Improvement: NUDT_CHINA 2019

Special Design

In order to improve this part, this 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). Due to the low efficiency of TATA box promoter, we shorten the sequence into only minip. The minip can be considered as a microcosm of TATA promoter, for its function is almost similar to TATA promoter.In addition, we also added glucose-sensing fragment to enhance the part’s initiation strength, as well as glucose-sensing function. Because there is high blood glucose concentration in diabetics, in order to get improved gene which is sensitive to diabetics it’s convenient to employ promoter which reacts to high blood glucose. 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 promoter of engineered plasmid and connect it with minP to indicates a transcription start site. In addition, we increase the number of CHoRE to realize that engineered gene will be activated by particular high blood glucose concentration instead of normal blood glucose concentration.
The structure diagram of the improved part is as below.

Figure 1. The structure diagram of the 9xGSP part.

Materials

PGL3-9XGSP

PGL3-miniP

Hepg2 cell line

Dual Luciferase Reporter Gene Assay Kit from from Beyotime company

pcdna3.1-9xgsp-gfp

Method

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 of 9xGSP using 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.Photograph makes the results clearer.

Figure 3.Transfection and Test of 9xGSP using GFP

Result

We chooose luciferase and GFP to report the experiment result respectively. When we use luciferase as reporting system.In the histogram below, the left is the result of PGL3-minP and the right is of PGL3-9XGSP. Taking renilla as the internal reference and comparing with the control experiment, we can find that the luciferase expression of PGL3-9XGSP is much higher than the expression of PGL3-minP. This result strong proves that CHoRE can significantly improve minP promoter’s promoting intensity.

Figure 4. The design and results of 9xGSP validation using luciferase.

When we use GFP as reporting system.After 18 hours’ transfection, we conduct experiments to test the function of our part. Photograph of fluorescence microscopy helps make results clear and obvious. Meanwhile, with the set of internal control, we can gain relative fluorescence intensity by Image J. During this process, we set different groups with different glucose concentration, which helps us to detect the relationship between GFP/mcherry and glucose concentration. Besides, test at different times makes the tendency of expression level as time passes much more clearer. From the figure we can easily discover that the glucose-sensing promoter can sense the glucose concentration and thus modify the expression level according to it. The figure below shows the results.

Figure 5. GFP/mcherry after 6 hours’ transfection(A). GFP/mcherry after 18 hours’ transfection(B). GFP/mcherry after 30 hours’ transfection(C). GFP/mcherry after 42 hours’ transfection(D). GFP/mcherry after 54 hours’ transfection(E).

Reference

[1] Li Ma,PengFei Gao,JianZhong Shi,et al.Research progress of ChREBP[J].Animal Husbandry and Feed Science,2014,35(09):40-42(Chinese)