Difference between revisions of "Part:BBa K337055"

Revision as of 09:29, 27 October 2010

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

characterization of part binding sites (BBa_K337055)

Binding sites for miR122 were experimentally characterized by cloning them into psiCHECK-2 backbone (Promega). [http://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay 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 (mut 99) 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.