Difference between revisions of "Team:BostonU/Design"

Latest revision as of 00:14, 19 September 2015

Overview

Our team demonstrated a prototype of our expected system - being able to conditionally dimerize a protein for genetic manipulation purposes. We investigate this more closely here using our split TP901-1 dimerization prototype.

Using the MATLAB model, we identified several promising split sites, and cloned these candidate splits into different conditional dimerization domain backbones. These domains would theoretically bind in the presence of the corresponding inducer, allowing the protein to regain functionality and demonstrate activity. In the absence of the inducer, the domains would not not dimerize, and the protein would theoretically be non-functional.

We used flow cytometry to measure the viability and efficiency of various candidate TP901-1 splits acting on a fluorescent reporter, and were delighted to see that some of our candidate split sites showed the expected results. This indicated that our split TP901-1 constructs were not functional in the absence of the inducer, dimerized in the presence of the inducer, and successfully catalyzed inversion to activate expression of a fluorescent protein. Below is one of our flow cytometry results validating this system:

The above TP901-1 integrase was split between amino acids 326-327, using FKBP-FRB dimerization domains. Addition of the rapalog inducer resulted in dimerization of the protein, leading to mRuby fluorescence.

Not only did we characterize functional splits, but we also validated functionality of multiple conditional dimerization backbones. Below is one of our flow cytometry results validating this:

The above TP901-1 integrase had the same split between amino acids 326-237, using ABI-PYL dimerization domains. Addition of the abscisic acid inducer resulted in dimerization of the protein, leading to mRuby fluorescence.

Ultimately, we have shown characterization of a conditional dimerization TP901-1 prototype. Using our methodology, we were able to predict viable splits and validate other conditional dimerization systems as well. TP901-1 has never been conditionally dimerized before, so our prototype is the first of its kind. Additionally, characterization of our systems in mammalian cells demonstrates successful operation in the chassis of interest. Ultimately, we have shown characterization of a conditional dimerization TP901-1 prototype. Using our methodology, we were able to predict viable splits and validate other conditional dimerization systems as well. TP901-1 has never been conditionally dimerized before, so our prototype is the first of its kind. Additionally, characterization of our systems in mammalian cells demonstrates successful operation in the chassis of interest.

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 <h3>Overview</h3>
 <p>
-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.
+Our team demonstrated a prototype of our expected system - being able to conditionally dimerize a protein for genetic manipulation purposes. We investigate this more closely here using our split TP901-1 dimerization prototype.
 </p>
 <p>
-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 is a viable TP901-1 split in the FKBP-FRB-Rapalog system.</p>
+Using the MATLAB model, we identified several promising split sites, and cloned these candidate splits into different conditional dimerization domain backbones. These domains would theoretically bind in the presence of the corresponding inducer, allowing the protein to regain functionality and demonstrate activity. In the absence of the inducer, the domains would not not dimerize, and the protein would theoretically be non-functional.</p>
+<p>We used flow cytometry to measure the viability and efficiency of various candidate TP901-1 splits acting on a fluorescent reporter, and were delighted to see that some of our candidate split sites showed the expected results. This indicated that our split TP901-1 constructs were not functional in the absence of the inducer, dimerized in the presence of the inducer, and successfully catalyzed inversion to activate expression of a fluorescent protein. Below is one of our flow cytometry results validating this system:</p>
 <br>
 <center><img style="height:50%; width:50%;" src="https://static.igem.org/mediawiki/2015/thumb/5/5d/Rap_tp901_.png/800px-Rap_tp901_.png" /><center>
 <br>
-<p>The TP901-1 integrase was split between amino acid 326 and 327. Addition of the rapalog inducer caused dimerization of the protein, leading to mRuby fluorescence</p>
+<p>The above TP901-1 integrase was split between amino acids 326-327, using FKBP-FRB dimerization domains. Addition of the rapalog inducer resulted in dimerization of the protein, leading to mRuby fluorescence.</p>
 <p>
-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. Below is the same split site in the ABI-PYL-ABA system.
+Not only did we characterize functional splits, but we also validated functionality of multiple conditional dimerization backbones. Below is one of our flow cytometry results validating this:
 </p>
 <br>
 <center><img style="height:50%; width:50%;" src="https://static.igem.org/mediawiki/2015/thumb/7/73/Aba_tp901_.png/800px-Aba_tp901_.png" /><center>
 <br>
-<p>These graphs show that we have characterized a split TP901-1 prototype between amino acids 326 and 327. We were able regain some protein activity after inducing dimerization of the split protein halves.</p>
+<p>The above TP901-1 integrase had the same split between amino acids 326-237, using ABI-PYL dimerization domains. Addition of the abscisic acid inducer resulted in dimerization of the protein, leading to mRuby fluorescence.</p>
 <p>
-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.
+Ultimately, we have shown characterization of a conditional dimerization TP901-1 prototype. Using our methodology, we were able to predict viable splits and validate other conditional dimerization systems as well. TP901-1 has never been conditionally dimerized before, so our prototype is the first of its kind. Additionally, characterization of our systems in mammalian cells demonstrates successful operation in the chassis of interest.
-</p>
+Ultimately, we have shown characterization of a conditional dimerization TP901-1 prototype. Using our methodology, we were able to predict viable splits and validate other conditional dimerization systems as well. TP901-1 has never been conditionally dimerized before, so our prototype is the first of its kind. Additionally, characterization of our systems in mammalian cells demonstrates successful operation in the chassis of interest.</p>
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