Difference between revisions of "Part:BBa K337057"
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| + | === characterization of part binding sites ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K337055 BBa_K337055]) === | ||
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| + | 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. [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337055 BBa_K337055] (mut 99) is the construct with the perfect binding site and leads to an knock-down percentage of 96%. [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337056 BBa_K337056] (mut 7) is an imperfect binding site which leads to a knockdown percentage of 64%. [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337057 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. | ||
| + | [[Image:PsiCheck.png|thumb|center|600px|'''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.]] | ||
Revision as of 09:30, 27 October 2010
synthetic binding site of miR 122 (imperfect) KD: 24%
imperfect binding site of miR122 in the tuning construct leads to a knockdown percentage of 64% percent if introduced into the 3'UTR of the gene of interest (GOI). part can be either co-transfected with a synthetic miRNA or transfected into liver cell lines (i.e. Huh7).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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). [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.
