Difference between revisions of "Part:BBa K2889003:Design"

Revision as of 02:53, 9 October 2018

pSB1C3-miR-21-sponge-4s

Design Notes

In our previous project, we constructed two miR-21 sponge plasmid, pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s, as the monitor to detect the expression of miR-21 in cells.The slope of the standard curve of miR-21-sponge-2s is better than miR-21-sponge-6s, suggesting the more sensitive of miR-21-sponge-2s as a monitor. This year, we wanted to improve the sensitive of miRNA sponge. We designed miR-21 sponges contains four miR-21 binding sites with 3-nt spacers for bulged sites based on the sequence of hsa-miR-21 according to the previous study.We constructed pEGFP-miR-21-sponge-4s and pSB1C3-miR-21-sponge-4s this year. Our data suggested miR-21-sponge-4s is more sensitive than miR-21-sponge-2s and miR-21-sponge-6s.

Source

We synthesized miR-21-sponge-4s from BBa_K2514000.

First 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.

We digested the PSB1C3 vectors with restriction enzymes EcoRI and PstI

https://parts.igem.org/File:MiR-21-sponge-4s_sequencing_map.jpeg

miR-21-sponge-4s fragments were ligated to PSB1C3 vector. Then we selected the positive clones by PCR and sequencing

References

Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007 Sep;4(9):721-6