Team:BostonU/Design

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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.