Part:BBa_K3064026
9xGSP-GFP
This composite part is made up of a kind of improved promoter which is sensitive to particular high blood glucose concentration and EGFP sequence. The introduction of EGFP 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
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. 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]
Characterization
This part BBa_K3064026 was cloned in pcDNA3.1+ 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 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
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.
Figure 3. 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).
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