Part:BBa_K2923018:Experience

Introduction

To validate the designed constructs we decided to test two different aptazymes: Theophylline switch-off (stop auto-cleavage in presence of ligand) and Guanine switch-on (active 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_K2923018

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).

Figure 1: Aptazyme catalytic activity assay. Theophylline aptazyme were transcribed in vitro for 1h30. RNA was analyzed in an 8% polyacrylamide gel with urea. The inactive form of Theophylline aptazyme represents the whole RNA and corresponds to the band of 94 pb. Cleaved aptazyme represents the two parts of the aptazyme Theophylline (18 pb + 76 pb).

In-vitro assay show, that PP7-PP7-Theophylline-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 empty plasmid backbone.

Theophylline aptazyme is switch-off (self-cleaves in absence of ligand) and prevents ß-galactosidase transcription. It was analyze in presence of its inactive form and BH2, BH3 controls (Figure 2).

Figure 2: ß-galactosidase assay on Theophylline aptazyme in absence of ligand

Theophylline aptazyme has an ß-galactosidase activity profile similar to BH2 and BH3 controls, witch promotes its use as a ligand sensor in the designed three-hybrid system. This data confirms that aptazymes could be compatible with three-hybrid systems.

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

For investigating theophylline aptazyme in vivo activity we choose concentration recommended of Theophylline ligand by Felletti et al., 2016: 2,5 mM. We performed the ß-galactosidase assay in the presence of its negative control and BH2/BH3 controls incubated with the same concentrations of theophylline (Figure 3).

Figure 3. ß-galactosidase assay on Theophylline aptazyme in presence of Theophylline (2,5 mM)

We obtained the expected results for all controls, so theophylline ligand did not have effects on the three-bacterial hybrid system. Despite that, the active form of theophylline aptazyme is not sensitive to its ligand. Any significant ß-galactosidase activity increase was observed in the presence of Theophylline ligand compared to an inactive control.

To increase the chances to obtain positives results for Theophylline aptazyme we choose to performed the same experiment with higher concentration of Theophylline ligand (5mM) (Figure 4).

Figure 4. ß-galactosidase assay on Theophylline aptazyme in presence of Theophylline (5 mM)

However, the obtained results shows that Theophylline do not respond on ligand presence.

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