Part:BBa_K2923020:Experience

Introduction

To validate the designed constructs we decided to test two different aptazymes Guanine switch-on (active auto-cleavage in the 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_K2923020

In order to check MS2-MS2-Guanine-PP7-PP7 cleavage activities, in-vitro transcription products were analyzed by 2% agarose gel with urea (Figure 1).

Figure 1: Aptazyme catalytic activity assay. Guanine aptazyme were transcribed in vitro for 1h30. RNA was analyzed in an 8% polyacrylamide gel with urea. The inactive form of Guanine aptazyme represents the whole RNA and corresponds to the band of 138 pb. Cleaved aptazyme represents the two parts of the aptazyme Guanine (28 pb + 110 pb).

In-vitro assay show, that PP7-PP7-Guanine-MS2-MS2 constructs have kept 100% of their ribozyme activity. Also, inactive aptazyme did not present aptazyme catalytic activity, which confirms their use as efficient negative control.

In-vivo: ß-galactosidase assay in the absence of ligand

Before to start an in vivo experiment on aptazymes, we characterized the constructs created by Berry KE and Hochschild as positive and negative controls. A ß-galactosidase activity positive control also called BH3 corresponds to BBa_K2923000. A ß-galactosidase activity negative control, called BH2 correspond to an empty plasmid backbone.

Guanine aptazyme is switch-on (self-cleaves in presence of ligand) and actives ß-galactosidase transcription. It was analyzed in the presence of its inactive form and BH2, BH3 controls (Figure 2).

Figure 2: ß-galactosidase assay on Guanine aptazyme in absence of ligand n=3

None expected results were observed for Guanine active, also then inactive aptazyme. It does not have significant ß-galactosidase activity levels compared to BH3 control.

In-vivo: ß-galactosidase assay in the presence of ligand

For investigating Guanine aptazyme in vivo activity we choose concentration recommended of Guanosine ligand by Felletti et al., 2016: 1 mM. We performed the ß-galactosidase assay in the presence of its negative control and BH2/BH3 controls incubated with the same concentrations of guanosine (Figure 3).

Figure 3. ß-galactosidase assay on Guanine aptazyme in presence of Guanosine (1 mM)

Any significant ß-galactosidase activity increase was observed in the presence of guanosine ligand compared to BH2/BH3 control. The active form of guanine aptazyme is not sensitive to its ligand. Also, then its inactive form is not compatible with BH3 system.

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