Latest revision as of 03:11, 22 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

This year we have made a series of modification and functional improvements based on the Minimum TATA-box promoter designed by Daniel Tang of Stanford BIOE44 - S11.(BBa_M50098). Therefore, quantitative experimental characterization were performed to characterize this mini promoter.In addition, we also added glucose-sensing fragment to enhance the part’s activation strength under the high glucose concentration. Hyperglycemia is a common symptom in Type two diabetic mellitus (T2D). In order to design a gene circuit that could ease symptoms of T2D automatically, sensing the high concentration of of blood glucose would be a essential and initial step of our GCGR degradation system. Thus, we improved the minimum TATA-box promoter to a novel functional part that could respond to hyperglycemia and activate transciption of our degradation system based on an existing Part(BBa_M50098). Technically,a major glucose responsive transcription factor -- ChREBP would be dephosphorylated under high blood glucose. the dephosphorylated ChREBP would subsequently enter the nucleus to activate the gene expression of genes containing Carbohydrate-responsive element (ChoRE) sequence¹.Therefore, we design a novel promoter contains several ChREBP binding sites and a basic mini promoter.To enable robust ChREBP binding among different species, we integrated previously reported ChREBP ChIP-Seq data in both human and mouse to obtain reserved binding motif. Motif enrichment analysis provided us a minimum sequence of CHREBP binding site. Hence, we reasoned that a glucose sensitive transcriptional activation can be achieved by repeating such binding motif several times upstream of the minimum promoter. This part were designed to respond to glucose concentration by repeating ChoRE sqeuence nine times upstream of mini promoter, termed as 9X glucose-sensing promoter(9X GSP). The structure diagram of the improved part is as below.

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

Materials

PGL3-9X GSP

PGL3-miniP

HepG2 cell line

Dual Luciferase Reporter Gene Assay Kit from Beyotime company

pcDNA3.1-9X GSP-gfp

Method

First, luciferase were chosen as the reporting system to quantitatively characterize the transcriptional strength of 9X GSP. HepG2 cells were transfected with PGL3-9X GSP and PGL3-minP, separately. To validate the glucose responsiveness, we challenged the 9x GSP-luciferase carrying HepG2 cells by culturing cells in different concentration of glucose after overnight starvation. After 48 hours, cells were harvested and Dual Luciferase Reporter Gene assay were performed to measure the level of firefly luciferase transcribed by 9X GSP.

Figure 2. Characterization of 9X GSP using firefly luciferase

To further characterize the glucose sensing module, we generated a 9X GSP-GFP reporter plasmid to increase the detection throughput. By using CMV-mcherry to normalize the effect of glucose on general exogenous gene expression level. Specifically, HepG2 cells were starved overnight with glucose-free culture and stimulated with 20mM glucose concentration for 6 or more hours. Flurosecent images were subsequently obtained and analyzed by ImageJ softwar.

Figure 3.Characterization of 9X GSP using GFP

Result

Luciferase and GFP were independently utilized as the reporter gene to characterize 9X GSP. In luciferase-based characterization, SV40 promoter-renilla luciferase were utilized to normalize the effect of glucose on general exogenous gene expression level. The results indicated that luciferase expression activiated by 9X GSP is significantly higher than the expression activited by mini promoter. This result strongly proved that CHoRE can significantly improve mini promoter's transciptional strength under the high glucose concentration (20mM).

Figure 4. The improvement of mini promoter and functional validation of 9X GSP based on luciferase.

In fluorescence protein-based characterization,CMV promoter-mcherry were used as internal control to normalize the effect of glucose on general exogenous gene expression level. At different time (6h, 18h, 30h, 42h, 54h) after transfection, fluorescence images were obtained by fluorescence microscope. Thus, fluorescence intensity of GFP could directly indicate the expression level of GFP under different glucose concentrations, which stands for transcriptional strength of 9X GSP. Results showed that 9X GSP is capable of activating gene expression in a glucose dose-dependent manner.
The figure below shows the characterization results.

Figure 5. 9X GSP activites GFP expression in a glucose dose-dependent manner. Fluorescence images were obtained by fluorescence microscope and fluorescence intensity of GFP or RFP(mcherry) was calculated via calculating the integral value of grayscale intensity in RGB chunnel respectively. the intensity of mcherry were used to normalize the GFP expression. (A)The GFP/mcherry value 6 hours after transfection. (B) The GFP/mcherry value 18 hours after transfection. (C) The GFP/mcherry value 30 hours after transfection.(D)The GFP/mcherry value 42 hours after transfection. (E)The GFP/mcherry value 54 hours after transfection.

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)