Difference between revisions of "Part:BBa K3160009:Design"
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===Design Notes=== | ===Design Notes=== | ||
The pEGFP-miR-196a sensorsensor was controlled by CMV promoter (CMV-GFP- miR-196a). The plasmids of CMV-GFP- miR-196a were transfected into different cell lines (one is human gastric epithelial cell line. The others are gastric cancer cell lines).We also measuresd the fluorescence of GFP in different cells transfected with CMV-GFP- miR-196a for 24 h by plate reader (SpectraMax i3), which analyses the potential value of CMV-GFP- miR-196a to measure the expression of miR-196a. | The pEGFP-miR-196a sensorsensor was controlled by CMV promoter (CMV-GFP- miR-196a). The plasmids of CMV-GFP- miR-196a were transfected into different cell lines (one is human gastric epithelial cell line. The others are gastric cancer cell lines).We also measuresd the fluorescence of GFP in different cells transfected with CMV-GFP- miR-196a for 24 h by plate reader (SpectraMax i3), which analyses the potential value of CMV-GFP- miR-196a to measure the expression of miR-196a. | ||
| + | 1．The effect of pEGFP-miR196a sensor in gastric cancer cells | ||
| + | The expression of miR-196a is highly associated with the early stage of gastric cancer [1]. So we constructed the plasmid [pEGFP-miR-196a sensor (kkb website)] and try to test the possibility of detecting tumor cells by using the plasmid. To detect the validity of pEGFP-miR196a sensor in cells, pEGFP–C1 (as negative controls), pEGFP-miR-196a sensor (0.8 ug plasmids for each well) was transfected into human gastric epithelial cells (GES-1 cells) in 24-well plate, respectively. After transfection, cells were examined under fluorescence microscopy (Fig. 1 A, B). The fluorescence of GFP was decreased in GES-1 cells transfected with pEGFP-miR-196a sensor or pEGFP-miR-196a-148a sensor compared with controls (Fig. 1 A, B, and C). Because pEGFP-miR-196a sensor contains binding sites of miR-196a and pEGFP-miR-196a-148a sensor contains binding sites of two miRNAs, the fluorescence of GFP was further decreased in GES-1 cells transfected with pEGFP-miR-196a-148a sensor compared with cells transfected with pEGFP-miR-196a sensor (Fig. 1 and table 1). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3). The endogenous miR-196a and miR-48a could inhibit the expression of GFP in cells transfected with pEGFP-miR-196a sensor or pEGFP-miR-196a-148a sensor (Table 1 and Fig 2). The similar results were also observed in two gastric cancer cells (SGC-7901 and MGC-803) (Fig 1; Fig 2 and Table 1). The result suggested pEGFP-miR-196a-148a sensor can show the different expression of miRNAs in cells. | ||
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| + | https://static.igem.org/mediawiki/parts/4/43/T--XHD-WS-Wuhan-A--196a_fig_1.jpeg | ||
===Source=== | ===Source=== | ||
Revision as of 11:14, 17 October 2019
pEGFP-miR-196a sensor
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The pEGFP-miR-196a sensorsensor was controlled by CMV promoter (CMV-GFP- miR-196a). The plasmids of CMV-GFP- miR-196a were transfected into different cell lines (one is human gastric epithelial cell line. The others are gastric cancer cell lines).We also measuresd the fluorescence of GFP in different cells transfected with CMV-GFP- miR-196a for 24 h by plate reader (SpectraMax i3), which analyses the potential value of CMV-GFP- miR-196a to measure the expression of miR-196a. 1．The effect of pEGFP-miR196a sensor in gastric cancer cells
The expression of miR-196a is highly associated with the early stage of gastric cancer [1]. So we constructed the plasmid [pEGFP-miR-196a sensor (kkb website)] and try to test the possibility of detecting tumor cells by using the plasmid. To detect the validity of pEGFP-miR196a sensor in cells, pEGFP–C1 (as negative controls), pEGFP-miR-196a sensor (0.8 ug plasmids for each well) was transfected into human gastric epithelial cells (GES-1 cells) in 24-well plate, respectively. After transfection, cells were examined under fluorescence microscopy (Fig. 1 A, B). The fluorescence of GFP was decreased in GES-1 cells transfected with pEGFP-miR-196a sensor or pEGFP-miR-196a-148a sensor compared with controls (Fig. 1 A, B, and C). Because pEGFP-miR-196a sensor contains binding sites of miR-196a and pEGFP-miR-196a-148a sensor contains binding sites of two miRNAs, the fluorescence of GFP was further decreased in GES-1 cells transfected with pEGFP-miR-196a-148a sensor compared with cells transfected with pEGFP-miR-196a sensor (Fig. 1 and table 1). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3). The endogenous miR-196a and miR-48a could inhibit the expression of GFP in cells transfected with pEGFP-miR-196a sensor or pEGFP-miR-196a-148a sensor (Table 1 and Fig 2). The similar results were also observed in two gastric cancer cells (SGC-7901 and MGC-803) (Fig 1; Fig 2 and Table 1). The result suggested pEGFP-miR-196a-148a sensor can show the different expression of miRNAs in cells.
Source
We ordered this part DNA from a synthesis company
References
1. Zheng G, Xiong Y, Xu W, Wang Y, Chen F, Wang Z, Yan Z. A two-microRNA signature as a potential biomarker for early gastric cancer. Oncol Lett. 2014 Mar;7(3):679-684.