Part:BBa_K3064014
CHoRE
It is a binding site of CHREBP.
Usage and Biology
Some glycolytic and lipogenic genes respond primarily to glucose, not to insulin. This led to the identification of a consensus sequence that is required for glucose-responsiveness, termed the carbohydrate response element (ChoRE), comprising of two E-boxes (CACGTG) or E-box-like sequences separated by 5 bp [3-5].
Carbohydrate response element binding protein (ChREBP) is an important transcription that can regulate the expression of key genes involved in various pathways including glycolysis, gluconeogenesis and lipogenesis. It does this by forming a tetrameric complex made up of two ChREBP/Mlx heterodimers, which enables it to bind to the carbohydrate response element (ChoRE) in the promoter region. ChoRE should be widely applicable to studying the influence of glucose consentration to cells .
Figure 1: ChREBP bind with ChoRE
The basic part BBa_K3064000 is an important part in our next experiment. The different number of CHoRE are set in other parts. And the part BBa_K3064026 shows his function. This part BBa_K3064026 was cloned in pGL3 and transfected into HepG2 cell lines using Invitrogen LipofectamineTM 3000. Protocol could be found in our Experiment page.
<ahref="https://2019.igem.org/wiki/images/1/1b/T--NUDT_CHINA--Protocol_for_lipo3000_transfection_with_Lipofectamine%E2%84%A2_3000_Reagent.pdf">Click to see </ a>
To conduct the later function test, we set five different groups to conduct the transfection. They are pGL3-9xGSP(glucogon sensor promoter)-GFP , pGL3-6xGSP-GFP, pGL3-3xGSP-GFP, pGL3-minip-GFPand 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Test
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. First, we set different groups with different promoter so that we can understand that the number of CHoRE can influence the promoter's efficiency. And we know that the more CHoRE has, the better effect we have. Then, we set the experiment that 6xGsp-GFP in different glucose concentration for 48 hours'. And we get the result that the effect is different in different glucose concentration.
Figure 2: Some structures and results on CHoRE
Figure 2 (A): Some strutures on CHoRE. Figure 2 (B): minip/3xGsp/6xGsp/9xGsp after 48 hours' transfection in 10 um glucose stimulation. Figure 2 (C): 6xGsp in different glucose concentration after 48 hours.
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