Part:BBa_K3822005

miR196a-148a 2.0

miR196a-148a 2.0 is an improvement of Part BBa_K3160007. which could monitor the expression of miRNAs in gastric cancer cells

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

1.Construction of the plasmids

The miRNA “ sponge” method was introduced by several years ago as a means to create continuous miRNA loss of function in cell lines and transgenic organisms. More effective are sponges containing bulged sites that are mispaired opposite miRNA positions 9-12 (Ebert et al. 2007, Gentner et al. 2009), presumably because they form a more stable interaction with the miRNA. pEGFP-miR196a-148a 2.0(Part K3822005) was designed based on the principle of effective sponges containing bulged sites (Fig. 1). We compare pEGFP-miR196a-148a 2.0 with pEGFP-miR196a-148a 1.0 (Part K3160007,designed by a 2019 iGEM team) on monitoring the expression of miRNAs in gastric cancer cells.

Fig 1. Pairing of a miRNA with a bulged sponge site shows mismatches opposite miRNA nucleotides.

The plasmids of pEGFP-miR-196a-148a 2.0 was synthesized by Nanjing Qingke Biotechnology Corporation. The electrophoresis of the plasmids was showed in Fig 2.

Fig 2. Electrophoresis of pEGFP-miR-196a-148a 2.0.

2. The effect of pEGFP-miR-196a-148a 1.0 and pEGFP-miR-196a-148a 2.0 in gastric cancer cells

To detect the validity of pEGFP-miR-196a-148a 1.0 and pEGFP-miR196a-148a 2.0 in cells, pEGFP–C1 (as negative controls), pEGFP-miR-196a-148a 1.0 or pEGFP-miR-196a-148a 2.0 (0.5 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 3A-C). The fluorescence of GFP was decreased in GES-1 cells transfected with pEGFP-miR-196a-148a 1.0 and pEGFP-miR-196a-148a 2.0 compared with controls (Fig 3 A-C). Because pEGFP-miR-196a-148a 2.0 contains more effective binding sites than pEGFP-miR-196a-148a 1.0, the fluorescence of GFP was further decreased in GES-1 cells transfected with pEGFP-miR-196a-148a 2.0 compared with that in cells transfected with pEGFP-miR-196a-148a 1.0 (Fig. 3 and Table 1). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3) (Fig. 3-4 and Table. 1). The similar results were also observed in SGC-7901 cells (gastric cancer cell line) (Fig. 3-4, and Table 1). Compared with pEGFP-miR-196a-148a 1.0, pEGFP-miR-196a-148a 2.0 is more effective to be inhibited by the endogenous miRNAs (Fig. 3 and Table. 1).

Fig 3. The images of different gastric cells transfected with different plasmids. (A). pEGFP–C1 (control) was transfected in GES-1 cells. (B). pEGFP-miR-196a-148a 1.0 was transfected in GES-1 cells. (C). pEGFP-miR-196a-148a 2.0 was transfected in GES-1 cells. (D). pEGFP–C1 (control) was transfected in SGC-7901 cells. (E). pEGFP-miR-196a-148a sensor 1.0 was transfected in SGC-7901 cells. (F). pEGFP-miR-196a-148a sensor 2.0 was transfected in SGC-7901 cells.