Team:TU-Eindhoven/Results/Orthogonality

iGEM TU Eindhoven

Orthogonality
Orthogonality with respect to wildtype interaction

The most important aspect of the mutation set is orthogonality with respect to the wildtype interaction. To test this, five measurements were compared to each other. The first one was a wildtype CT52–SmallBiT and a mutated CT52-LargeBiT. For an orthogonal heterodimer this should give the highest interaction between scaffold and binding partner.

The second measurement was two mutated CT52 proteins, each linked to a part of the NanoBiT system. This should not give a high binding interaction when assembled on a heterodimer, because the mutated CT52 protein should not have high affinity for a wildtype T14-3-3 binding pocket.

The third measurement is the same as the second measurement, except that this measurement is done with a wildtype CT52 proteins linked to each NanoBiT. This should also give a low activity because wildtype CT52 should not have high affinity for mutated T14-3-3 binding pockets.

The fourth and fifth measurement are a measurement without heterodimer present and a measurement without heterodimer and CT52 present, respectively. These should also yield low values. This data was visualized by integrating the measured bioluminescence over time. This is visualized in bar graphs.

T14-3-3(S71L) and CT52(I947H)

T14-3-3(S71L) and CT52(I947H) Mutation set T14-3-3(S71L) and CT52(I947H) showed the expected curve in the functionality test. In figure 1 is shown that this mutation set is not completely orthogonal, but the first measurement, with the theoretical best combination of CT52-LargeBiT and CT52-SmallBiT show activity about two times higher compared to the other measurements. Based on these measurements it can be concluded that this mutation is partially orthogonal. The activity which was expected to be the highest is significantly higher so this can be a useful mutatation.

Figure 1: 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.
T14-3-3(S71L-I72V) and CT52(I947F)

Mutation set T14-3-3(S71L&I72V) and CT52(I947F) showed the expected curve in the functionality test. In figure 2, it is shown that this mutation set is not completely orthogonal. Even though the first measurement shows activity about two times higher compared to the other measurements, the standard deviations are rather high which makes it unreliable. Based on these measurements it can be concluded that this mutation is can also be partially orthogonal, however with less certainty than the previous mutation set.

Figure 2: T14-3-3 orthogonality measurement with 1 µM T14-3-3(W237R)-T14-3-3 for bar 1-3, 0 µM T14-3-3(W237R)-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(S953K)-LargeBiT,CT52-SmallBiT. Bar 2: CT52(S953K)-LargeBiT,CT52(S953K)-SmallBiT. Bar 3: CT52(wildtype)-LargeBiT,CT52(wildtype)-SmallBiT. Bar 4: CT52(S953K)-LargeBiT,CT52(wildtype)-SmallBiT. Bar 5: No CT52-LargeBiT or CT52-SmallBiT present. The substrate used was 1250x diluted from stock.
T14-3-3(W237R) and CT52(S953K)

Mutation set T14-3-3(W237R) and CT52(S953K) did not show the expected curve in the functionality test. The activity was very low compared to the other heterodimers. In figure 3 below is visible that this mutation set is not orthogonal at all. There is high activity when only wildtype CT52-split NanoLuc present, indicating that there is a possibility that CT52(S953K) is not a stable protein, causing no binding interaction with the scaffold at all. Based on these measurements it can be concluded that this mutation is not orthogonal.

Figure 3: T14-3-3 orthogonality measurement with 1 µM T14-3-3(W237R)-T14-3-3 concentration for bar 1-3, 0 µM T14-3-3(W237R)-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(S953K)-LargeBiT,CT52(wildtype)-SmallBiT. Bar 2: CT52(S953K)-LargeBiT,CT52(S953K)-SmallBiT. Bar 3: CT52(wildtype)-LargeBiT,CT52(wildtype)-SmallBiT. Bar 4: CT52(S953K)-LargeBiT,CT52(wildtype)-SmallBiT. Bar 5: No CT52-LargeBiT or CT52-SmallBiT present. The substrate used was 1250x diluted from stock.
Control mutation set: T14-3-3(E19R),CT52(K943D)

The control mutation set T14-3-3(E19R),CT52(K943D) kind of showed the expected curve in the functionality test. In figure 4 below is visible that the CT52(K943D) is partially orthogonal with respect to the wildtype T14-3-3. The graph also suggests that wildtype CT52 is not able to bind to the mutated scaffold. However this is a quite remarkable result, since the activity is even lower than the negative control.

Figure 4: T14-3-3 orthogonality measurement with 1 µM T14-3-3(W237R)-T14-3-3 concentration for bar 1-3, 0 µM T14-3-3(W237R)-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(K943D)-LargeBiT,CT52(wildtype)-SmallBiT. Bar 2: CT52(K943D)-LargeBiT,CT52(K943D)-SmallBiT. Bar 3: CT52(wildtype)-LargeBiT,CT52(wildtype)-SmallBiT. Bar 4: CT52(K943D)-LargeBiT,CT52-SmallBiT. Bar 5: No CT52-LargeBiT or CT52-SmallBiT present. The substrate used was 1250x diluted from stock.
Orthogonality with respect to other mutation sets

For each mutation set its orthogonality with respect to the wildtype binding interaction has been determined (figure 5). Secondly, the orthogonality between each mutation set is useful in the evaluation of each mutation and for future purposes when some mutation sets might be combined. But this property is less important for our project, since our main goal is to create a mutation set which is orthogonal with respect to the wildtype interaction.

Figure 5: this table shows an overview of each mutation set and orthogonality with respect to each other. To illustrate how to read this table an example will be given: For the scaffold with a S71L mutation this table gives information about how well each CT52 variant binds to it. The CT52 S943K is not able to bind this is represented with dark red, but the CT52 K943D will bind weakly, because it is weakly orthogonal.

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