Difference between revisions of "Team:BostonU/App 1/Design"
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<p>After looking into the literature, our team decided to split two integrases: TP901-1 and PhiC31, and their corresponding RDFs: orf7 and gp3 (respectively). These integrase-RDF pairs can recognize orthogonal sites, and therefore can be used alongside each other in a robust system.</p> | <p>After looking into the literature, our team decided to split two integrases: TP901-1 and PhiC31, and their corresponding RDFs: orf7 and gp3 (respectively). These integrase-RDF pairs can recognize orthogonal sites, and therefore can be used alongside each other in a robust system.</p> | ||
| − | <center><img style="height:25%; width:25%;" src="https://static.igem.org/mediawiki/2015/thumb/5/54/Integrase_RDF_wo_inducer.png/800px-Integrase_RDF_wo_inducer.png" /><nbp><nbp><nbp><img style="height:25%; width:25%;" src="https://static.igem.org/mediawiki/2015/thumb/7/71/Integrase_RDF_RDF_w_inducer.png/800px-Integrase_RDF_RDF_w_inducer.png" /><nbp><nbp><nbp | + | <center><img style="height:25%; width:25%;" src="https://static.igem.org/mediawiki/2015/thumb/5/54/Integrase_RDF_wo_inducer.png/800px-Integrase_RDF_wo_inducer.png" /><nbp><nbp><nbp><img style="height:25%; width:25%;" src="https://static.igem.org/mediawiki/2015/thumb/7/71/Integrase_RDF_RDF_w_inducer.png/800px-Integrase_RDF_RDF_w_inducer.png" /><nbp><nbp><nbp><center> |
<p>We applied our split protein pipeline [link to this] to determine promising split locations for both integrases and RDFs. We chose 8 candidate integrase split sites and 4 candidate RDF split sites, and cloned these into our mammalian expression plasmid backbones that included fusion to our sets of dimerizable domains. We independently tested the functionality of these conditionally dimerizable proteins using fluorescent reporter plasmids.</p> | <p>We applied our split protein pipeline [link to this] to determine promising split locations for both integrases and RDFs. We chose 8 candidate integrase split sites and 4 candidate RDF split sites, and cloned these into our mammalian expression plasmid backbones that included fusion to our sets of dimerizable domains. We independently tested the functionality of these conditionally dimerizable proteins using fluorescent reporter plasmids.</p> | ||
<p>For testing the functionality of our conditionally dimerizable integrases, we used a fluorescent reporter containing an inverted mRuby coding sequence flanked by two recombination sites that could be specifically recognized by the particular integrase (i.e. we used a reporter with TP901-1 recognizable sites for the split TP901-1 constructs, and a reporter with PhiC31 recognizable sites for the split PhiC31 constructs). We co-transfected this reporter with our pairs of plasmids that coded for our conditionally dimerizable split proteins, and assayed for fluorescent activity in the absence and presence of the drug. We expected that the “OFF” state (absence of inducer), there was no mRuby expression, whereas in the “ON” state (presence of inducer) there was mRuby expression.</p> | <p>For testing the functionality of our conditionally dimerizable integrases, we used a fluorescent reporter containing an inverted mRuby coding sequence flanked by two recombination sites that could be specifically recognized by the particular integrase (i.e. we used a reporter with TP901-1 recognizable sites for the split TP901-1 constructs, and a reporter with PhiC31 recognizable sites for the split PhiC31 constructs). We co-transfected this reporter with our pairs of plasmids that coded for our conditionally dimerizable split proteins, and assayed for fluorescent activity in the absence and presence of the drug. We expected that the “OFF” state (absence of inducer), there was no mRuby expression, whereas in the “ON” state (presence of inducer) there was mRuby expression.</p> | ||
| − | <center><img style="height:25%; width:25%;" src="https://static.igem.org/mediawiki/2015/thumb/c/c2/Integrase_RDF_RDF_flipping.png/789px-Integrase_RDF_RDF_flipping.png" /><center> | + | <center><img style="height:25%; width:25%;" src="https://static.igem.org/mediawiki/2015/thumb/c/c2/Integrase_RDF_RDF_flipping.png/789px-Integrase_RDF_RDF_flipping.png" /><nbp><nbp><nbp><img style="height:25%; width:25%;" src="https://static.igem.org/mediawiki/2015/thumb/6/61/Integrase_flipping.png/800px-Integrase_flipping.png" /><center> |
<p>For testing the functionality of our conditionally dimerizable RDFs, we used a similar fluorescent reporter containing an inverted iRFP coding sequence flanked by two recombination sites that could be specifically recognized by the particular RDF (i.e. we used a reporter with TP901-1+orf7 recognizable sites for the split orf7 constructs, and a reporter with PhiC31+gp3 recognizable sites for the split gp3 constructs). We co-transfected this reporter with our pairs of plasmids that coded for our conditionally dimerizable split proteins as well <i>as with a plasmid that coded for the corresponding full integrase</i>, and assayed for fluorescent activity in the absence and presence of the drug. We expected that the “OFF” state (absence of inducer), there was no iRFP expression, whereas in the “ON” state (presence of inducer) there was iRFP expression.</p> | <p>For testing the functionality of our conditionally dimerizable RDFs, we used a similar fluorescent reporter containing an inverted iRFP coding sequence flanked by two recombination sites that could be specifically recognized by the particular RDF (i.e. we used a reporter with TP901-1+orf7 recognizable sites for the split orf7 constructs, and a reporter with PhiC31+gp3 recognizable sites for the split gp3 constructs). We co-transfected this reporter with our pairs of plasmids that coded for our conditionally dimerizable split proteins as well <i>as with a plasmid that coded for the corresponding full integrase</i>, and assayed for fluorescent activity in the absence and presence of the drug. We expected that the “OFF” state (absence of inducer), there was no iRFP expression, whereas in the “ON” state (presence of inducer) there was iRFP expression.</p> | ||
Revision as of 19:52, 18 September 2015
| Motivation | Design | Results |
Design
After looking into the literature, our team decided to split two integrases: TP901-1 and PhiC31, and their corresponding RDFs: orf7 and gp3 (respectively). These integrase-RDF pairs can recognize orthogonal sites, and therefore can be used alongside each other in a robust system.
We applied our split protein pipeline [link to this] to determine promising split locations for both integrases and RDFs. We chose 8 candidate integrase split sites and 4 candidate RDF split sites, and cloned these into our mammalian expression plasmid backbones that included fusion to our sets of dimerizable domains. We independently tested the functionality of these conditionally dimerizable proteins using fluorescent reporter plasmids.
For testing the functionality of our conditionally dimerizable integrases, we used a fluorescent reporter containing an inverted mRuby coding sequence flanked by two recombination sites that could be specifically recognized by the particular integrase (i.e. we used a reporter with TP901-1 recognizable sites for the split TP901-1 constructs, and a reporter with PhiC31 recognizable sites for the split PhiC31 constructs). We co-transfected this reporter with our pairs of plasmids that coded for our conditionally dimerizable split proteins, and assayed for fluorescent activity in the absence and presence of the drug. We expected that the “OFF” state (absence of inducer), there was no mRuby expression, whereas in the “ON” state (presence of inducer) there was mRuby expression.
For testing the functionality of our conditionally dimerizable RDFs, we used a similar fluorescent reporter containing an inverted iRFP coding sequence flanked by two recombination sites that could be specifically recognized by the particular RDF (i.e. we used a reporter with TP901-1+orf7 recognizable sites for the split orf7 constructs, and a reporter with PhiC31+gp3 recognizable sites for the split gp3 constructs). We co-transfected this reporter with our pairs of plasmids that coded for our conditionally dimerizable split proteins as well as with a plasmid that coded for the corresponding full integrase, and assayed for fluorescent activity in the absence and presence of the drug. We expected that the “OFF” state (absence of inducer), there was no iRFP expression, whereas in the “ON” state (presence of inducer) there was iRFP expression.
We also carried out necessary controls for each experiment, including testing our individual fluorescent proteins for proper detection, and our fluorescent reporters without any split integrases co-transfected. Our most important control was to test the activity of the full, un-split protein relative to each of the split protein pairs. In order to quantitatively assess how active our conditionally dimerizable proteins were relative to our un-split proteins, we used the following equation:
