Difference between revisions of "Part:BBa K3064026"

Revision as of 03:27, 22 October 2019

9xGSP-GFP

This composite part is made up of a kind of improved promoter which is sensitive to particular high blood glucose concentration and GFP sequence. The introduction of GFP makes it possible to examine the expression of this composite part. As designed,the part can be used as a glucose-sensing promoter with GFP reporter system.

Usage and Biology

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 degradation system. Thus, we design a new 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.
Sequence and Features

Characterization

This part BBa_K3064026 was cloned in pcDNA3.1+ plasmid and transfected into HepG2 cell lines using Invitrogen LipofectamineTM 3000. Protocol could be found in our Experiment page.Click https://2019.igem.org/wiki/images/1/1b/T--NUDT_CHINA--Protocol_for_lipo3000_transfection_with_Lipofectamine%E2%84%A2_3000_Reagent.pdf to see detail information.
To conduct the later function test, we set two different groups to conduct the transfection. One is pcDNA3.1-9xGSP-GFP and the other one is plv-mcherry as the internal control. We transfected 300μg into HepG2 cells, which were cultured on 24-hole plate. When 90 percent were mixed, we begin the transfection. 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 of 48h.

Figure 1. Steps of transfection and function test.

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 minp. In addition, we also added glucose-sensing fragment to enhance the part’s initiation strength, as well as glucose-sensing function. With GFP, the part’s function can be better detected.

Figure 2. The structure diagram of the 9xGSP-GFP part.

Function Test

Fluorescence protein-based characterization were performed to test the glucose-sensing function of promoter,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 3. 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)