- Policy & Practices
We designed, expressed and tested three sets of mutations and on this page they are evaluated on performance. They are evaluated on two criteria, namely functionality and orthogonality. However, in order to draw definite conclusions additional testing will be necessary.
Let’s start with mutation set T14-3-3(S71L) and CT52(I947H). This mutation set showed great promise. The relative intensity assay showed the greatest resemblance to the bioluminescence of the E19R mutation set, which is promising because the E19R mutation set functioned as a control. Also the assays with varying scaffold concentrations yielded a smooth increasing trend, which was exactly what we expected. This means that the affinity of CT52(I947H) for T14-3-3(S71L) still is great enough to bind. So this mutation set has good functionality. Next the orthogonality should be considered. The graph showed that the mutation set is not completely orthogonal, but the binding from wildtype CT52 to the mutated T14-3-3 and the binding between the mutated CT52 and wildtype T14-3-3 are considerably weaker than the complementary set. This is a positive result since creating a binding which is completely orthogonal is extremely challenging.
We also tested the mutation set T14-3-3(S71L&I72V) and CT52(I947F). This mutation set also had comparable results with respect to the E19R mutation set. Yet the trend seen in the assays with varied scaffold concentrations was not as smooth as we would have expected. On orthogonality it did score quite well, it is not completely orthogonal but this mutation is also very promising.
Lastly, the mutation set T14-3-3(W237R) and CT52(S943K) was also tested. This mutation set did not score great on functionality. Both graphs, relative intensity and varied scaffold concentrations, showed that the mutation set resulted in a dysfunctional system. From these graphs cannot be concluded if the mutation in the T14-3-3 or the mutation in the CT52 is responsible. The orthogonality assays have given some insights, but do suggest that the mutation in the CT52 is largely responsible for the disruption. However, the measured bioluminescence was so low a definite conclusion cannot be drawn. A conclusion that can be drawn is that this mutation set did not work.