Team:BostonU/App 1/Results

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Motivation

Design

Results

Results

When performing flow cytometry, we measured mRuby fluorescence values in arbitrary units. Each transfection condition was performed in triplicate on every plate, and thus we were able to obtain the mean and standard deviation across three replicates. In order to normalize the data, so we could compare values across different days of testing, the following equation was used:

Utilizing this equation, we could evaluate the activity of conditionally dimerized integrases and RDFs relative to the corresponding intact protein.

We normalized each of the values against the values of the respective intact protein control, took the mean and standard deviation of these values, and then plotted them. Using this method, the intact integrase control was set to 100% activity, and the relative activity of all splits was measured.

In all of our subsequent plots, we show percentage activity of each conditionally dimerized protein, both with and without the corresponding inducer (black vs. color bar respectively).

Our first success was to develop functional, conditionally dimerizable integrases and RDFs! We were able to create many functional split TP901-1 and PhiC31 candidates. Below is an example of one of our best TP901-1 split sites (between amino acids 326-327), using the FKBP-FRB dimerization domains.

We noticed that the overall activity of the conditionally dimerized protein in the presence of the Rapalog inducer was ~37% of the intact integrase. Furthermore, the activity of the conditionally dimerized protein in the absence of the inducer was ~2% of the intact integrase. While this was promising because the un-dimerized split had very low basal activity and high intact activity, we wondered how choosing other split sites, dimerization domains, and domain orientations could affect protein activities.

Some other questions that we identified included:

1. How does conditional dimerization of TP901-1 integrase compare to that of PhiC31 integrase?
2. Can we identify any patterns between split site locations and corresponding activity?
3. Do different dimerization domains affect efficiency of a particular split site?
4. Does the orientation of the dimerization domains affect the efficiency of a specific split site?

Below is an example comparing two split PhiC31 proteins (between amino acids 269-270 and 348-349), using the same FKBP-FRB dimerization domains.

Here, we saw that the split site between AA348-AA349 showed greater activity compared to AA269-AA270. We hypothesized that this may because AA348-AA349 is a split located between two different functional domains while AA269-AA270 is a split located within an annotated functional domain of the protein. Given this result, we would further analyze this hypothesis to confirm if the same trend occurred in other candidate split proteins, and if splitting between functional domains is always more efficient than within a functional domain.

Below is an example testing how a split TP901-1 integrase location (between amino acids 326-327) worked using ABI-PYL dimerization domains. We were interested in not only identifying activity of split proteins using this dimerization domain pair, but also how orientation of domains could affect activity.

From this result, we identified that the conditional dimerization split site showed more activity in one domain orientation compared to the reverse domain orientation. We were really interested in this result! We would like to further characterize how the same split locations work in different domain orientations, and if one orientation works better across all splits or different orientations are more or less effective in different splits.

One of the most exciting parts of our project related to testing efficiency of different dimerization domains was to test the CRY2-CIBN blue light inducible domains. Unfortunately, we had a lot of trouble developing a device that would emit blue light to stimulate dimerization. We decided to order a string of blue LED lights and construct our own device, but we ended up receiving white LED lights. We attempted to induce our CRY2-CIBN light constructs using these white LED lights anyways, and ran our experiment. Below is an example of our tested TP901-1 split construct using CRY2-CIBN domains.

Our results did not look particularly promising for this domain, and we think it is because our blue light induction system was not optimal. A better system would provide stronger blue light stimulus and have equal induction for each transfection.

Ultimately, we compared how both different domain pairs and orientations of domains affected functionality of the same split site. Below is an example of our tested split TP901-1 integrase (between amino acids 326-327) using both FKBP-FRB and ABI-PYL domains in both orientations.

We noticed that in this experiment, the FKBP-FRB domains had higher activity when induced compared to ABI-PYL domains, regardless of orientation. While, the ABI-PYL domains had lower basal activity in the absence of the inducer. This reinforces the idea that the larger dimerization domains hindered protein function more. However, more splits will have to be tested in both domains to conclude that this is the case for most proteins and split sites. In addition, we saw that for both dimerization domains, one particular orientation gave higher activity than the other.

These results were all obtained for the split integrase proteins, and we attempted to test our conditionally dimerizable RDF proteins as well. However, we had less success cloning and testing these dimerization pairs. We were only able to run one round of flow cytometry for these constructs.

For gp3 RDF we were only able to test one split configuration. Below is the result of our split gp3 (between AA31-AA32) compared to the intact gp3 control.

We anticipated that something was not optimal in this transfection experiment, as we noticed extremely low activity for the intact gp3 control but much higher activity for the split gp3 variant. We concluded that repeating the experiment would be the only way to further explore this result.

For the orf7 RDF we did not successfully clone any conditionally dimerizable variants. We did validate that our intact orf7 control functioned against our reporter. Below is the activity of the intact orf7, which we ultimately submitted to the iGEM registry.