Difference between revisions of "Part:BBa K3201004"

Revision as of 22:25, 21 October 2019

S.mansoni Type 1 Hammerhead Ribozyme, Inactive

This is part BBa_K3201002, but with a mutation inserted in its active center so it is depleted of its ability to cleave itself.

Usage and Biology

This is the original Hammerhead ribozyme (HHR) from S. mansoni, mutated so that it cannot cleave itself. We have computationally modelled the molecule to determine its structure, which is shown in Figure 1. The original HHR (without mutations) is also shown in Figure 2.

Figure 1: Inactive HHR from S. mansoni.

Figure 2: (Active) HHR from S. mansoni.

As it can be seen, the two differ significantly. Therefore, we expected that the inactive HHR would not be as efficient as the original HHR at self-cleavage.

We, therefore, assessed its catalytic activity through an in vitro HHR cleavage assay. First, we produced sufficient amounts of the ribozyme through in vitro transcription. We incorporated a T7 promoter and used the in vitro transcription kit from NEB (T2050S). The in vitro transcription products were run in a 1% agarose gel and the result is shown in Figure 2.

Figure 3: In vitro transcribed inactive HHR. Lane 1: Ladder 50 bp (NEB). Lane 2: In vitro transcription positive control (NEB). Lane 3: In vitro transcribed original HHR. Lane 4: In vitro transcribed inactive HHR

Then we prepared the in vitro cleavage assay. In an eppendorf tube the following were added: 0.5 μL of in vitro transcribed HHR, 0.5 μL of 1 M Tris-HCl (final concentration 50 mM), 0.3 μL of 1 M MgCl2 (final concentration 30 mM), and 8.7 μL of nuclease-free water. The mixture was incubated in an incubator at 37 degrees Celsius for 1 hour. After 1 hour, the reaction was stopped using 10 μL of 6X loading dye. The mixture was then heated at 70 degrees Celsius for 5 minutes and then loaded on a gradient (4-20%) polyacrylamide gel. Before electrophoresis, the gel was prerun for 30 mins at 150V. The PAGE was done using 1X TBE as the running buffer and at a constant voltage of 150 V. The gel was stained overnight in Ethidium Bromide. The result is shown in Figure 3.

Figure 4: In vitro cleavage assay.

As it can be seen, the inactive HHR is not as efficient at self-cleavage as the original HHR and almost no product is observed. Therefore, the inactive HHR can be used as a negative control or as a tool in synthetic circuits.

Sequence and Features