GUPPI is a system to induce gelation. In the last few years, quite some papers have been published about gelation and liquid-liquid phase separation. [1-4] The consensus throughout literature is that gelation can be achieved by using protein structures that can undergo multiple interactions. These proteins are known as multivalent proteins. The influence of multivalency on cluster formation has been evaluated in both intra and extra cellular applications. This is shown by both experimental and in silico work. [5-7]

Based on this information, we have designed a novel system based on a well studied protein-protein interaction. In our project, we have created two multivalent constructs that interact with each other to form a network of proteins, the basics of a gel. More information about the principles and proteins can be found in the "Background".

The System

Our designed system is named GUPPI, after Gelation Using Protein Protein Interactions, see also "Design & Parts".
Just as the iGEM team before us, we will use the 14-3-3 protein as basis of our designed system. Last year, the 14-3-3 protein was used as scaffold and they mutated many different sites to tune the interaction strength. This year we will go back to one type of 14-3-3, and instead of mutating sites of the monomer, we will use it as building block for a very large protein, consisting out of multiple monomers. The 14-3-3 protein naturally forms a dimer with itself, and thus has a valency of 2. We increased the amount of monomers via DNA design, which led to a valency of 4. However, studies [5-7] have shown that a valency ratio of 3:4 shows promising results in creating gel structures. Therefor a simple DNA mutation in the last monomer of the construct has been performed, we blocked the functionality of the last monomer, leading to a functional valency of 3. The structure of this construct is shown schematically in Figure 1.


Figure 1: Mutation of the last 14-3-3 pocket to make it non-functional

The second construct needed for our project is the protein CT33. CT33 is a H(+)-ATPase derivative and can bind into the pocket of a 14-3-3 monomer. This interaction is relatively weak, but can be increased excessively by adding a small molecule, fusicoccin, that acts as a stabilizer. This means that CT33 is a very nice basis for a construct to combine with 14-3-3 for forming a network after inducing the system with fusicoccin. As the C-terminus of CT33 is necessary for the correct folding of the protein, it was not possible to design one protein out of multiple CT33 parts. The solution we found is to use a Strep-tag® II together with Strep-Tactin®XT. Strep-tag® II is mostly known for its application in protein purification, just like a His-tag. The Strep-tag® system is based on the strongest non-covalent interaction known in nature, the interaction of biotin with streptavidin. The Strep-tag®II consists of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). This peptide tags exhibit high affinities to the resins Strep-Tactin® and Strep-Tactin® XT which are specifically engineered streptavidins. Strep-Tactin®XT is a protein with four binding sites for Strep-tag® II. We used Strep-Tactin®XT to create a construct consisting out of four CT33 proteins, leading to our second construct, which has thus a valency of 4. The CT33 construct is schematically shown in Figure 2 below.


Figure 2: Assembly of the CT33 construct with Strep-Tactin®XT to get a valency of 4

When combining these two constructs in presence of an inducer, network formation can occur. The network formation is depicted schematically in Figure 3.

Figure 3: Interactions of 14-3-3 with CT33 and Strep-tag® II with Strep-Tactin®XT cause network formation

[1] S. F. Banani, H. O. Lee, A. A. Hyman, and M. K. Rosen, “Biomolecular condensates: organizers of cellular biochemistry,” Nat. Rev. Mol. Cell Biol., vol 18, pp 285-298 2017.
[2] S. Banjade and M. K. Rosen, “Phase transitions of multivalent proteins can promote clustering of membrane receptors”, eLife, 2014
[3] S. F. Banani, A. M. Rice, W. B. Peeples, Y. Lin, S. Jain, R. Parker, M. K. Rosen, S. F. Banani, A. M. Rice, W. B. Peeples, Y. Lin, S. Jain, R. Parker, and M. K. Rosen, “Compositional Control of Phase-Separated Cellular Bodies,” Cell, vol. 166, pp. 651–663, 2016.
[4] P. Li, S. Banjade, H.C. Cheng, B. Chen, L. Guo, M. Llaguno, J.V. Hollingsworth, D.S. King, S.F. Banani, P.S. Russo, Q.X. Jiang, B.T. Nixon and M.K. Rosen, “Phase transitions in the assembly of multivalent signalling proteins”, Nature, vol. 483, pp 336-340, 2012.
[5] T. Z. Grove, C. O. Osuji, J. D. Forster, E. R. Dufresne, and L. Regan, “Stimuli-Responsive Smart Gels Realized via Modular Protein Design,” J. Am. Chem. Soc., vol. 132, pp. 14024–14026, 2010.
[6] T. Z. Grove, J. Forster, G. Pimienta, E. Dufresne, and L. Regan, “A Modular Approach to the Design of Protein-Based Smart Gels A Modular Approach to the Design of Protein-Based Smart Gels,” Biopolymers, vol. 97, no. 7, pp. 508–517, 2012.
[7] C. T. S. Wong, P. Foo, J. Seok, W. Mulyasasmita, A. Parisi-amon, and S. C. Heilshorn, “Two-component protein-engineered physical hydrogels for cell encapsulation,” Proc. national Acad. Sci. United States Am., vol. 106, no. 52, pp. 22067–22072, 2009.