Part:BBa_K2923019:Experience
Introduction
To validate the designed constructs we decided to test Theophylline switch-off (stop auto-cleavage in presence of ligand). For negative control, we choose an inactive form of aptazyme: mutation at the ribozyme domain. iGEM Strasbourg designed construct requires the use of aptazyme linked to MS2 and PP7 stem-loops. The following results described MS2-MS2-Aptazyme-PP7-PP7 activity in in-vitro and in-vivo assays.
In-vitro assay of BBa_K2923019
In order to check MS2-MS2-Theophylline-PP7-PP7 cleavage activities, in-vitro transcription products were analyzed by 2% agarose gel with urea (Figure 1).
Inactive aptazyme did not present aptazyme catalytic activity, which confirms their use as an efficient negative control.
To see in-vivo assays results of MS2-MS2-Theophylline-PP7-PP7 aptazymes see BBa_K2923018.
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