We constructed two plasmids pEGFP-miR-21-sponge-4s and PSB1C3-miR-21-sponge-4s. The plasmids of pEGFP-miR-21-sponge-4s and PSB1C3-miR-21-sponge-4s were used to monitor the expression of miR-21 in cells. And the plasmid of pSB1C3-miR-21-sponge-4s will be submitted to 2018IGEM.
Amplification of miR-21-sponge-4s fragment.
To monitor the expression level of miR-21 in different cells, we made plasmid of miR-21 sponges in the 3’UTR of GFP. Firstly, we designed miR-21 sponges contains four miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-miR-21-sponge-4s) based on the sequence of hsa-miR-21 according to the previous study. Then we amplified miR-21-sponge-4s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoR I and Pst I (Fig 4).
(Fig 1. Electrophoresis of miR-21-sponge-4s PCR product)
Digested PSB1C3 vector(Fig 1).
Ligation of purified miR-21-sponge-4s fragments to PSB1C3 vector.miR-21-sponge-4s fragments were ligated to PSB1C3 vector. Then we selected the positive clones by PCR and sequencing (Fig 2).
(Fig2. miR-21-sponge-4s sequencing map)