Description: In the emerging field of synthetic biology, many new innovations arise. To use them as efficient and safe as possible, regulation is the key. Therefore, iGEM TU Eindhoven is developing new kinds of scaffold proteins, based on the T14-3-3 protein. These scaffold proteins have a wide range of applications in regulating systems in synthetic biology. 14-3-3 proteins are a protein family which are highly conserved and are expressed in all eukaryotic cells. We are working with 14-3-3 proteins from the Nicotiana plumbaginifolia (Tobacco) plant (T14-3-3). T14-3-3 proteins dimerize to form a functional scaffold. A T14-3-3 scaffold is known to interact with the last 52 C-terminal amino acids of the regulatory domain of tobacco H+-ATPase (CT52)1. It is also known that this interaction is greatly stabilized by the natural product Fusicoccin2. Thus under influence of FC two CT52 protein can bind to the T14-3-3 scaffold. The first scaffold protein that we will make is a heterodimeric variant of the T14-3-3 protein. A second orthogonal binding interaction between T14-3-3 and CT52 compared to the wildtype needs to be found to create a heterodimer. This possible new binding interaction was found through computational design, using the Rosetta software package. With this New scaffold protein, one could bring two different proteins in close proximity of each other. One of the possible applications of this protein is regulating the CRISPR/Cas9 system. In a recent study from Zetsche, Volz, & Zhang3, the authors successfully split the Cas9 protein into two inactive fragments which can assemble into active Cas9 when in close proximity of each other. When each of these two sCas9 fragments is linked to the CT52 protein, they could be assembled on our scaffold protein under the influence of fusicoccin, creating a novel switchable CRISPR/Cas9 system that can be regulated with fusicoccin. The second protein that will be made by our team is a tetrameric variant of the T14-3-3 protein. This will be accomplished by linking four 14-3-3 monomers to each other. It is expected this protein will have a stronger response with respect to the naturally occurring dimeric variant of T14-3-3, because a larger amount of CT52 can bind in comparison with the dimeric variant. An application of this protein is a kill switch. To make a functional kill switch, caspase 9 is used, a protein which induces apoptosis when brought in close proximity of each other. By linking caspase 9 to CT52, apoptosis can be induced dependent on the presence of fusicoccin, thus a kill switch is created. This kill switch can be implemented in genetically modified cells as an extra safety mechanism. An example of implementation of the kill switch is T cell therapy. This is an emerging possible cancer treatment in which human T cells are extracted from the body, then they are genetically modified to detect and exterminate cancer cells more efficient when placed back in the body of the patient.4 Our kill switch might be valuable to ensure the safety of this treatment. This is not expected to happen on short term, it is just one example of the possibilities our kill switch might have in the future.
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Year: 2016Visit Wiki
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Updated at: 8/9/16