Team:Athens/Improve
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MEDEA/Improve ΜΕΔΕΑ
Improve
Part Improvement
A STAR is (re)born
During the development of our project we had to decide on a messenger that would activate our selection system when the aptazyme would bind to our target molecule. At first, we were leaning towards the implementation of a genetic XNOR gate logic from Bonnett et al., 2013, [1] which would be inactivated by the cleaved part of the aptazyme, a siRNA. However, this solution proved to be too complicated and difficult to be incorporated in a chaotic biological system.
A possible solution emerged when we stumbled upon the Imperial College 2016 iGEM team wiki, who proposed a novel small RNA category as a synthetic biology tool: the Small Transcription Activating RNA.[2] You can read more about how we adapted this technology into our Design.
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 our beloved 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
In order to demonstrate our improvement, we evaluated our STAR - HHRz complex in two ways: in silico and in vivo.
In silico evaluation
Firstly we began by visualizing the conjugation between part BBa_K1893013 and our Hammerhead Ribozyme, to ensure that the addition did not disrupt the stereometry of the HHRz. Using RNAfold and RNAComposer, we deduced that it did not cause any interference, as you can see in the visual below, created with Chimera:
However, we realised that a significant portion of the HHR is also cleaved during the process, therefore increasing the length of the STAR sequence by a few bases. We decided to alter our STAR sequence, by referencing to the sequences created computationally by the team that invented the STAR system [3] and choosing the one that had the same 3’ sequence as the 5’ sequence of the HHRz. This way, there would be no additional bases left. We then compared the DNA strand displacement rates for the initial and our optimized sequence:
Therefore we had a good indication that our design would be functional in vivo.
Experimental evaluation
We then characterised the STAR - HHRz joined molecule both in vitro and in vivo. At first, we performed an in vitro Hammerhead Activation Assay using MgCl2 and tested the product of the assay using PAGE electrophoresis. This showed that our design was still functional. You can find more on the assay on our Results page.
We therefore deduce that our HHR-compatible STAR can be implemented in RNA engineering, with minimal efficiency losses. The conjugation of HHRs and STARs offers a new avenue to manipulate biological systems. Through the incorporation of aptamers in the system it is possible to make new cellular sensors that activate transcription in the presence of molecules recognised by the aptamer. Moreover, the HHR-STAR conjugation could be used in cell-free systems to control transcription and its products.
1. Bonnet, J., Yin, P., Ortiz, M. E., Subsoontorn, P. & Endy, D. Amplifying Genetic Logic Gates. Science 340, 599–603 (2013).
2. iGEM Imperial College 2016, Basic Parts
3. Chappell, J., Westbrook, A., Verosloff, M. & Lucks, J. B. Computational design of small transcription activating RNAs for versatile and dynamic gene regulation. Nature Communications 8, 1051 (2017).