Team:TU-Eindhoven/Proof

iGEM TU Eindhoven

Proof of Concept

In the beginning of this project mutation sets were designed using the Rosetta software package. With these designs it was possible for us to create a possible functional heterodimeric T14-3-3 protein. This theoretical prediction was executed in the lab. As described on the Functionality and Orthogonality pages, it was concluded that 2 out of the 3 designed mutation sets were working as a (partly) orthogonal scaffold.

To show a proof of concept, one mutation set is highlighted, namely the mutation set with mutation S71L in the T14-3-3 domain and mutation I947H in the CT52 domain. More information on these mutations can be found here.

The two important characteristics of a successful mutation set were determined:

  • Functionality of the scaffold protein, representing the ability of the scaffold protein to bind with its complementary binding partner. In case of T14-3-3(S71L) this should be the ability to bind to CT52(I947H).
  • Orthogonality of the designed mutation set. The mutated binding pocket,T14-3-3(S71L) should only have a strong binding interaction with its complementary binding partner,CT52(I947H). This also works vice versa: CT52(I947H) should only have a strong binding interaction with its complementary binding pockets, which is T14-3-3(S71L).

The functionality of the heterodimeric T14-3-3(S71L) was tested in vitro with the NanoBiT reporter system by adding wildtype CT52-SmallBiT and CT52(I947H)-LargeBiT to the scaffold. It was concluded that mutation set T14-3-3 (S71L) / CT52 (I947H) meets the first criterion.

Figure 1: T14-3-3 functionality measurement with varying scaffold concentrations from 0 nM to 1 µM T14-3-3(S71L)-T14-3-3. 50 µM fusicoccin was used for all measurements. Mutant forms CT52 (I947H)-LargeBiT and CT52(wildtype)-SmallBiT were used with a concentration of 200 nM. The substrate used was 1250x diluted from stock.

The orthogonality of the mutation set was also tested with the NanoBiT reporter system by five measurements. In the first measurement one wildtype CT52-SmallBiT and one CT52(I947H)-LargeBit were added to the scaffold. For an orthogonal heterodimer this should give the highest interaction. In the second measurement two CT52(I947H) proteins linked to SmallBiT and LargeBiT were added to the T14-3-3(S71L) scaffold. This should not give a high activity, because CT52(I947H) protein should not have affinity for the wildtype T14-3-3 scaffold monomer. The third measurement is similar to the second measurement, except that this measurement was performed two wildtype CT52 proteins. This should also yield a low activity because wildtype CT52 should not have high affinity for the T14-3-3(S71L) scaffold monomer. The fourth and fifth measurement were measurements without heterodimer present and without both heterodimer and CT52 present. These should also yield a low activity, because the activity of the first measurement is significantly higher compared to the other measurements. Therefore, it is concluded that the mutation set T14-3-3 (S71L) / CT52 (I947H) also passes for criterion 2.

Figure 2: T14-3-3 orthogonality measurement with 1 µM T14-3-3(S71L)-T14-3-3 for bar 1-3, 0 µM T14-3-3(S71L)-T14-3-3 for bar 4 and 5. 50 µM fusicoccin for bar 1 -5. Mutant forms CT52-LargeBiT and CT52-SmallBiT are present in different combinations. Bar 1: CT52(I947H)-LargeBiT & CT52(wildtype)-SmallBiT, bar 2: CT52(I947H)-LargeBiT & CT52(I947H)-SmallBiT, bar 3: CT52-LargeBiT & CT52(wildtype)-SmallBiT, bar 4: CT52(I947H)-LargeBiT & CT52(wildtype)-SmallBiT and bar 5: No CT52-LargeBiT or CT52-SmallBiT present. The substrate used was 1250x diluted from stock.

The mutation set T14-3-3 (S71L) / CT52 (I947H) meets both criteria and is therefore a successful mutation.

Conclusion

This shows our team has successfully translated a theoretical model in a system that works in vitro. The research performed by iGEM TU Eindhoven has proven that this mutation itself is a working concept and can now for example be used for complex chemical conversions that are performed in vitro, in which our scaffold enables the formation of a system which regenerates the coenzymes. This results in a reduction of costs and allowing the use of enzymes in complex chemical produces processes, as described our application scenarios.

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