Regulatory

Part:BBa_K337055

Designed by: Rebecca Berrens   Group: iGEM10_Heidelberg   (2010-10-26)

synthetic binding site of shRNA 122 (perfect) KD96:%

perfect binding site of liver specific shRNA 122 in the context of the tuning construct. This binding side leads to a knockdown percentage of 96% percent if introduced into the 3'UTR of the gene of interest (GOI). It can be either co-transfected with a synthetic miRNA or transfected into liver cell lines (i.e. Huh7).

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]


characterization of part binding sites (BBa_K337055)

Binding sites for miR122 were experimentally characterized by cloning them into psiCHECK-2 backbone (Promega). Dual luciferase assay was conducted using this time Renilla Luciferase as a reporter and the other luciferase as a reference for normalization. Figure 2 shows again a broad range of regulation depending on binding site sequence properties. BBa_K337055 (perfect BS) is the construct with the perfect binding site and leads to an knock-down percentage of 96%. BBa_K337056 (mut 7) is an imperfect binding site which leads to a knockdown percentage of 64%. BBa_K337057(mut 111) is an imperfect binding site with a knockdown efficiency of 24%. All this contributes to a real tuning effect by introducing binding sites with introduced mismatches following the rational design protocol.

Figure 3: Tuning of gene expression through different imperfect miR122 binding sites in psiCHECK-2. Construct was transfected into HeLa cells together with an plasmid expressing miR122. Control without binding site was used for normalization.
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