Part:BBa_K3201003
S.mansoni Type 1 Hammerhead Ribozyme with STAR module
This is part BBa_K3201002 conjugated with the STAR part
Usage and Biology
During our project, we aimed to improve a previous part the small-transcription activating RNA first proposed by iGEM ICL 2016 (https://parts.igem.org/Part:BBa_K1893013). We observed that this tool could be further improved, by being connected to another RNA molecule which would control the release of the STAR molecule. This would be a hammerhead ribozyme molecule. The STAR molecule is only released once the hammerhead ribozyme has been self-cleaved. In this way, the transcriptional activation exerted by the STAR can be controlled and regulated. This molecule could also be utilized as an aptazyme. In this case, the STAR will be released only and only if the aptazyme binds to its specific ligand
Here we have deposited the the original Hammerhead ribozyme from S. mansoni conjugated to a small-transcription activating RNA (STAR). We have computationally modelled the molecule to determine its structure, which is shown in Figure 1. The structure can be compared to the original HHR structure, which is shown in Figure 2.
As it can be seen, the catalytic domain has not changed significantly and we therefore expected the HHR conjugated to STAR to function normally.
We 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.
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.
Successful cleavage results to the disappearance of a band at around 200 bp, which is the size of the HHR approximately (including its terminator). As shown, the conjugation doesn't influence the cleavage. Therefore, STARs and HHRs can be coupled together for tighter control of STAR activities.
We further constructed plasmids containing the STAR target molecule followed by RFP. These vectors carried either an inactive STAR, the HHR conjugated to a STAR, and the original STAR alone. In this way, red colour is produced when a successful interaction between the STAR and STAR target is done, which in the case of the HHR conjugated STAR would mean that the HHR can be cleaved also in vivo.
The inactive STAR showed only a few red colonies, as shown in the Figure below.
The original STAR alone yielded many red colonies.
The HHR-conjugated STAR yielded also many red colonies.
These results indicate that neither STAR nor HHR lose their functionality in vitro or in vivo when they are conjugated. This can potentially result to new ways of controlling gene expression.
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]
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