Difference between revisions of "Team:BostonU/Design"
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We used flow cytometry in order to test the viability of our split TP901-1 and we were delighted to validate some viable split sites. In the presence of an inducer, the protein halves dimerized and we were able to regain the TP901-1 integrase activity, validating these split sites as functional. TP901-1 catalyzes inversion reactions, and our flow cytometry experiment involved having our dimerized split TP901-1 flip an otherwise unexpressed fluorescent protein, leading to the expression of said fluorescent protein. Our results showed that there were indeed some TP901-1 splits that would lead to expression of this fluorescent reporter, and we were able to conclude that these split sites were viable, since we were able to regain the original activity of the TP901-1 protein. Below are some viable TP901-1 splits in the FKBP-FRB-Rapalog system. | We used flow cytometry in order to test the viability of our split TP901-1 and we were delighted to validate some viable split sites. In the presence of an inducer, the protein halves dimerized and we were able to regain the TP901-1 integrase activity, validating these split sites as functional. TP901-1 catalyzes inversion reactions, and our flow cytometry experiment involved having our dimerized split TP901-1 flip an otherwise unexpressed fluorescent protein, leading to the expression of said fluorescent protein. Our results showed that there were indeed some TP901-1 splits that would lead to expression of this fluorescent reporter, and we were able to conclude that these split sites were viable, since we were able to regain the original activity of the TP901-1 protein. Below are some viable TP901-1 splits in the FKBP-FRB-Rapalog system. | ||
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Revision as of 20:51, 18 September 2015
Overview
Our team was able to demonstrate a split TP901-1 protein prototype. Using the model developed in MATLAB, we were able to identify several different places to rationally split a protein. Both inert protein fragments were fused to dimerization domains, that naturally bind in the presence of an inducer. The protein showed low basal activity, and regained activity in the presence of the inducer.
We used flow cytometry in order to test the viability of our split TP901-1 and we were delighted to validate some viable split sites. In the presence of an inducer, the protein halves dimerized and we were able to regain the TP901-1 integrase activity, validating these split sites as functional. TP901-1 catalyzes inversion reactions, and our flow cytometry experiment involved having our dimerized split TP901-1 flip an otherwise unexpressed fluorescent protein, leading to the expression of said fluorescent protein. Our results showed that there were indeed some TP901-1 splits that would lead to expression of this fluorescent reporter, and we were able to conclude that these split sites were viable, since we were able to regain the original activity of the TP901-1 protein. Below are some viable TP901-1 splits in the FKBP-FRB-Rapalog system.
Not only were we able to characterize several different split sites as functional, but we also tested these split sites out with different inducers and in different orientations of dimerization domains. We further optimized our split TP901-1 prototype by finding the optimal conditions for which to create a successful split with robust activation activity.
While this split methodology has been applied to other proteins, TP901-1 has never been split before, so our prototype is the first of its kind. Our prototype also shows the feasibility of our model and our experimental design, and we realize that our workflow can also be extended to create other new split protein prototypes.