- Policy & Practices
In the emerging field of synthetic biology, which has a major impact on society, there have been many new innovations such as the CRISPR/Cas9 system and gene drives. Because the application of these brand new technologies can have irreversible effects on our surroundings, ensuring correct uses and especially safety is of utmost importance. We aim to improve these important aspects of synthetic biology by developing new protein tools: Regulatable scaffold proteins.
iGEM TU Eindhoven uses the T14-3-3 scaffold protein, which originates from the tobacco plant, as a base for their development of the regulatable scaffold. This is because it is known to have an interaction with the C-terminal end of H(+)-ATPase (CT52) that is greatly stabilized by the small molecule fusicoccin.1 The appealing thing about CT52 is that it has a free N-terminal end to which any protein can be attached via a flexible linker.2,3
The first goal of our team was to develop new orthogonal binding interactions between CT52 and T14-3-3. These new orthogonal binding interactions were found through computational design, using the Rosetta software package, resulting in three sets of mutations. Each newly found binding interaction was tested on functionality and orthogonality. The advantage of finding new mutations is that it makes the creation of new heterodimeric T14-3-3 scaffold proteins possible, on which two different CT52-protein complexes can assemble instead of two identical.
The second goal was to design a tetrameric variant of T14-3-3. With this tetramer it would be possible to bring four proteins together instead of two, because it contains four binding grooves. The tetramer was created by linking two T14-3-3 dimers together with a flexible linker. A tetrameric T14-3-3 scaffold could be used to create higher local concentration of protein compared to the homodimer T14-3-3 scaffold.
We also took one more step to combine our new orthogonal binding interactions with the tetramer and designed a tetramer with four different binding pockets, enabling the assembly of four different proteins. These scaffold proteins have a large variety of possible uses.
These possible uses vary from functioning as a kill switch in modified cells to optimizing processes in industry to regulating a bacterial drug delivery system for clinical uses and even regulation of the CRISPR/Cas9 system. To clarify the enormous amount of applications for our new scaffold proteins, three application scenarios were designed.
We have improved two biobricks from last year’s iGEM team from the TU Eindhoven, namely the two NanoBiT fragments called SmallBiT BBa_K1761006 and LargeBiT BBa_K1761005. We have expanded on their characterisation and application possibilities by linking it to our CT52 protein and documented our improvements on their main pages.
-  Ottmann, C., et al. (2007). Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+ -ATPase by combining X-ray crystallography and electron cryomicroscopy. Mol Cell, 25(3), 427-440.
-  Paiardini, A., Aducci, P., & Al, E. T. (2014). The phytotoxin fusicoccin differently regulates 14-3-3 proteins association to mode III targets. Life, 66(1), 52-62.
-  Skwarczynska, M., Molzan, M. and Ottmann, C. (2012). Activation of NF- B signalling by fusicoccin-induced dimerization. Proceedings of the National Academy of Sciences, 110(5), pp.E377-E386.