Team:uOttawa/project

From 2014.igem.org

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             <a href="#" data-pane="wrefs">References</a>
             <a href="#" data-pane="wrefs">References</a>
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<p id="desc-project">
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                Read an introduction about this year's project.
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            </p>
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<p id="desc-tristable">
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                We describe our approaches in creating the proposed tri-stable switch network.
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            </p>
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<p id="desc-promoters">
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                uOttawa designed a handful of complex and dual-input yeast promoters for this year's project. Read about them here.
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            </p>
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<p id="desc-wres">
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                See comparisons and characterization of each promoter.
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            </p>
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<p id="desc-bricks">
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                Self explanatory.
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            </p>
             <p id="desc-interlab">
             <p id="desc-interlab">
                 View uOttawa's results for the iGEM measurement track's Interlab Study.
                 View uOttawa's results for the iGEM measurement track's Interlab Study.
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            </p>
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<p id="desc-wrefs">
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                Want more info on the tristable switch and dual-input promoters? These papers are an excellent place to start.
             </p>
             </p>
         </nav>
         </nav>
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<figure>
<figure>
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                     <img src="https://static.igem.org/mediawiki/2014/5/55/Lake.jpg"">
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                     <img style="width:75%;" src="https://static.igem.org/mediawiki/2014/5/55/Lake.jpg"">
                 </figure>
                 </figure>
             </div>
             </div>
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                 <h2>Our designs</h2>
                 <h2>Our designs</h2>
                 <p>In order to implement this network, A and B appear to have to work as both an activator (to activate themselves) and a repressor (of the other state). Thus, we had to design a system where A and B can function as both. In brief, activator binding sites were placed 10bp away from the TATA box, causing steric hindrance of the TATA binding protein. This is explained in more depth, along with accompanied data in the <b>Promoters</b> section.</p>
                 <p>In order to implement this network, A and B appear to have to work as both an activator (to activate themselves) and a repressor (of the other state). Thus, we had to design a system where A and B can function as both. In brief, activator binding sites were placed 10bp away from the TATA box, causing steric hindrance of the TATA binding protein. This is explained in more depth, along with accompanied data in the <b>Promoters</b> section.</p>
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                 <p>For our system to work, two controllable transcription activators were selected, GEV and rTTA. GEV is a fusion protein of the gal4 DNA binding domain (G), a human estrogen receptor subunit (E), and VP16 (V), a viral trans-activator. To function, two GEV molecules must dimerize with a beta-estradiol molecule, localizing the GEV to the nucleus and allowing for activation. As such, how much GEV is active can be controlled via estradiol concentration. Similarly, rTTA is the tetR binding domain with a VP16 trans-activator, which requires anhydrotetracycline (aTc) in order to function. Thus, we can control how much of each trans-activator is active at any given time by varying the concentration of these small molecules. This is important for our design, as it allows us to test the functionality of our promoters, and examine our system from various 'start points' or states.</p>
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                 <p>For our system to work, two controllable transcription activators were selected, GEV and rtTA. GEV is a fusion protein of the gal4 DNA binding domain (G), a human estrogen receptor subunit (E), and VP16 (V), a viral trans-activator. To function, two GEV molecules must dimerize with a beta-estradiol molecule, localizing the GEV to the nucleus and allowing for activation. As such, how much GEV is active can be controlled via estradiol concentration. Similarly, rtTA is the tetR binding domain with a VP16 trans-activator, which requires anhydrotetracycline (aTc) in order to function. Thus, we can control how much of each trans-activator is active at any given time by varying the concentration of these small molecules. This is important for our design, as it allows us to test the functionality of our promoters, and examine our system from various 'start points' or states.</p>
                 <p>To create this system, we engineered two designs, both of which are based upon this core interaction:</p>
                 <p>To create this system, we engineered two designs, both of which are based upon this core interaction:</p>
                 <figure>
                 <figure>
                     <img src="https://static.igem.org/mediawiki/2014/1/10/Uo2014-wet6.png" alt="">
                     <img src="https://static.igem.org/mediawiki/2014/1/10/Uo2014-wet6.png" alt="">
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                     <p>Tri-stable involved portion of our network. Activation arrows are shown in green, and repression arrows in red. GEV and rTTA are transcriptional activators. pGALtx is a promoter activated by GEV and repressed by rTTA. pTREgx is a promoter activated by rTTA and repressed by GEV. tPGK1 is a transcriptional terminator.</p>
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                     <p>Tri-stable involved portion of our network. Activation arrows are shown in green, and repression arrows in red. GEV and rtTA are transcriptional activators. pGALtx is a promoter activated by GEV and repressed by rtTA. pTREgx is a promoter activated by rTTA and repressed by GEV. tPGK1 is a transcriptional terminator.</p>
                 </figure>
                 </figure>
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                 <p>Thus, in order to create this system, we had to design novel promoters to drive GEVE and rTTA, along with promoters to drive reports with similar expression levels. Their design, construction, and testing are described in the Promoters section</p>
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                 <p>Thus, in order to create this system, we had to design novel promoters to drive GEVE and rtTA, along with promoters to drive reports with similar expression levels. Their design, construction, and testing are described in the Promoters section</p>
                 <p>We then created two designs with this core.</p>
                 <p>We then created two designs with this core.</p>
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                 <figure>
                 <figure>
                     <img src="https://static.igem.org/mediawiki/2014/b/b6/Uo2014-network.png" alt="">
                     <img src="https://static.igem.org/mediawiki/2014/b/b6/Uo2014-network.png" alt="">
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                     <p>Constitutive promotion by the native yeast promoter mrp7 of each transcriptional activator. Gal U and Gal D represent DNA overhangs for transformation into the Gal4 locus, His is the histidine selection cassette, and tPGK1 is a transcriptional terminator.</p>
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                     <p>Constitutive promotion by the native yeast promoter pMRP7 of each transcriptional activator. Gal U and Gal D represent DNA overhangs for transformation into the Gal4 locus, His is the histidine selection cassette, and tPGK1 is a transcriptional terminator.</p>
                 </figure>
                 </figure>
                 <p>Unfortunately, we were unable to complete construction of either design due to problems with construction and transformation. However, we were able to test each functional part independently, so completion is now simply a matter of assembly.</p>
                 <p>Unfortunately, we were unable to complete construction of either design due to problems with construction and transformation. However, we were able to test each functional part independently, so completion is now simply a matter of assembly.</p>
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                 <p>All promoters were based upon the pGAL promoter found natively in Saccharomyces cerevisiae. Activating sites were placed where gal4 sites or mig1 sites already existed, in the hope of not disturbing the activity of this strong promoter. Repressing sites were placed 10 base pairs downstream of the TATA box, which was shown by Ellis et al. 2009 to retain promoter activity, as they placed repressing sites 10bp away from the TATA box.</p>
                 <p>All promoters were based upon the pGAL promoter found natively in Saccharomyces cerevisiae. Activating sites were placed where gal4 sites or mig1 sites already existed, in the hope of not disturbing the activity of this strong promoter. Repressing sites were placed 10 base pairs downstream of the TATA box, which was shown by Ellis et al. 2009 to retain promoter activity, as they placed repressing sites 10bp away from the TATA box.</p>
                 <h2>Repression by hindrance</h2>
                 <h2>Repression by hindrance</h2>
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                 <p>In short, transcription is repressed by sterically hindering the binding of the TATA binding protein to the TATA box. Thus, despite the bound proteins being an activator complex, transcription is prevented. Our data proves that this repression is not only strong and robust once activator concentration passes a certain saturation point. Activators only have a positive effect at very low concentrations. Along with very steep repression after a point, this indicates that this form of repression requires a certain saturation point before taking effect.</p>
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                 <p>In short, transcription is repressed by sterically hindering the binding of the TATA binding protein to the TATA box. Thus, despite the bound proteins being an activator complex, transcription is prevented. Our data proves that this repression is not only strong and robust once activator concentration passes a certain saturation point. Activators only have a positive effect at very low functional concentrations, and even then it is minute. Along with very steep repression after a point, this indicates that this form of repression requires a certain saturation point before taking effect.</p>
                 <figure>
                 <figure>
                     <img src="https://static.igem.org/mediawiki/2014/c/ca/Uo2014-wet4.png" alt="">
                     <img src="https://static.igem.org/mediawiki/2014/c/ca/Uo2014-wet4.png" alt="">
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                 <p>In cells expressing rtTA and GEV (GAL4 binding domain-human estrogen receptor-VP16 activator domain), this promoter can be used to drive transcription of a downstream gene by the addition of aTc (anhydrotetracycline). The level of transcription can be modulated or repressed with the addition of beta-estradiol.</p>
                 <p>In cells expressing rtTA and GEV (GAL4 binding domain-human estrogen receptor-VP16 activator domain), this promoter can be used to drive transcription of a downstream gene by the addition of aTc (anhydrotetracycline). The level of transcription can be modulated or repressed with the addition of beta-estradiol.</p>
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                 <h2>pTreVg</h2>
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                 <h2>pTre(4)</h2>
                 <figure>
                 <figure>
                     <img src="https://static.igem.org/mediawiki/2014/e/ea/Uo2014-prom2.png" alt="">
                     <img src="https://static.igem.org/mediawiki/2014/e/ea/Uo2014-prom2.png" alt="">
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                 <p>In cells expressing rtTA, this promoter can be used to drive transcription of a downstream gene by the addition of aTc (anhydrotetracycline).</p>
                 <p>In cells expressing rtTA, this promoter can be used to drive transcription of a downstream gene by the addition of aTc (anhydrotetracycline).</p>
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                 <h2>pTre</h2>
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                 <h2>pTre(2)</h2>
                 <figure>
                 <figure>
                     <img src="https://static.igem.org/mediawiki/2014/7/7e/Uo2014-prom3.png" alt="">
                     <img src="https://static.igem.org/mediawiki/2014/7/7e/Uo2014-prom3.png" alt="">
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                 <figure class="full">
                 <figure class="full">
                     <img src="https://static.igem.org/mediawiki/2014/b/bd/Uo2014-res2.png" alt="">
                     <img src="https://static.igem.org/mediawiki/2014/b/bd/Uo2014-res2.png" alt="">
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                     <p>Four activator site pGALtx under different repressor saturation. pGALtx has 4 activating gal4 sites and 2 repressing tet sites. On the left, rtTA is driven by the weak constitutive promoter mrp7 and on the right rtTA is driven by the strong constitutive promoter pADH1. aTc reprosents amount of repressing activator funcitonal, while estradiol activating activator.  </p>
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                     <p>Four activator site pGALtx under different repressor saturation. pGALtx has 4 activating gal4 sites and 2 repressing tet sites. On the left, rtTA is driven by the weak constitutive promoter pMRP7 and on the right rtTA is driven by the strong constitutive promoter pADH1. aTc represents the amount of functional repressing activator, while estradiol the amount of functional activating activator.  </p>
                 </figure>
                 </figure>
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   <figure>
   <figure>
                     <img src="https://static.igem.org/mediawiki/2014/e/ef/Uo2014-res4.png" alt="">
                     <img src="https://static.igem.org/mediawiki/2014/e/ef/Uo2014-res4.png" alt="">
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                     <p>Characterization of pGAL via dual drug induction. pGAL has no repressing sites and 4 activating gal4 sites, so increasing estradiol increases activation while aTc has no effect beyond auto-fluorescence. This particular data set has GEV being driven by mrp7, a week constitutive promoter.</p>
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                     <p>Characterization of pGAL via dual drug induction. pGAL has no repressing sites and 4 activating gal4 sites, so increasing estradiol increases activation while aTc has no effect beyond auto-fluorescence. This particular data set has GEV being driven by pMRP7, a week constitutive promoter.</p>
                 </figure>
                 </figure>
  <p><b> pTRE(4)gx </b></p>             
  <p><b> pTRE(4)gx </b></p>             
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   <figure>
   <figure>
                     <img src="https://static.igem.org/mediawiki/2014/1/17/Ptre2site.png"">
                     <img src="https://static.igem.org/mediawiki/2014/1/17/Ptre2site.png"">
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                     <p>Characterization of pTREgx via dual drug induction. pTREgx has 2 activating tetO sites and 2 repressing gal4 sites 10bp away from the TATA box. Thus, activation increases with aTc concentration and repression increases with estradiol concentration. Repression is minimal as GEV is only weakly promoted by mrp7, unlike the pTRE(4)gx data, where a stron promoter pADH1 is driving GEV </p>
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                     <p>Characterization of pTREgx via dual drug induction. pTREgx has 2 activating tetO sites and 2 repressing gal4 sites 10bp away from the TATA box. Thus, activation increases with aTc concentration and repression increases with estradiol concentration. Repression is minimal as GEV is only weakly promoted by pMRP7, unlike the pTRE(4)gx data, where a strong promoter pADH1 is driving GEV </p>
                 </figure>
                 </figure>
  <p><b> pTRE(4) </b></p>             
  <p><b> pTRE(4) </b></p>             
   <figure>
   <figure>
                     <img src="https://static.igem.org/mediawiki/2014/d/d3/PTRE_4_sites.png"">
                     <img src="https://static.igem.org/mediawiki/2014/d/d3/PTRE_4_sites.png"">
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                     <p>Characterization of pTRE via dual drug induction. pTRE has 4 activating tetO sites and not repressing sites, so increasing aTC increases activation. Estradiol has no effect beyond auto-fluorescence.  </p>
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                     <p>Characterization of pTRE via dual drug induction. pTRE has 4 activating tetO sites and not repressing sites, so increasing aTc increases activation. Estradiol has no effect beyond auto-fluorescence.  </p>
                 </figure>
                 </figure>
</figure>
</figure>
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   <figure>
   <figure>
                     <img src="https://static.igem.org/mediawiki/2014/3/3e/Ptre%282%29.png"">
                     <img src="https://static.igem.org/mediawiki/2014/3/3e/Ptre%282%29.png"">
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                     <p>(LEFT) The pTre promoter shows no activation upon addition of aTc (anhydrotetracycline)in cells expressing the activator, rtTA (reverse tetracycline-controlled transactivator), with the weak constitutive promoter, pMRP7. (RIGHT) In cells expressing rtTA with the strong constitutive promoter, pADH1, there is weak activation with increasing aTC concentration. Increasing concentrations of estradiol was added to compare its effects on activation with the other cognate promoters made. No increase in activation is seen with increasing estradiol due to the absence of GAL4 UAS's in this promoter. </p>
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                     <p>(LEFT) The pTre promoter shows no activation upon addition of aTc (anhydrotetracycline)in cells expressing the activator, rtTA (reverse tetracycline-controlled transactivator), with the weak constitutive promoter, pMRP7. (RIGHT) In cells expressing rtTA with the strong constitutive promoter, pADH1, there is weak activation with increasing aTc concentration. Increasing concentrations of estradiol was added to compare its effects on activation with the other cognate promoters made. No increase in activation is seen with increasing estradiol due to the absence of GAL4 UAS's in this promoter. </p>
                 </figure>
                 </figure>
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<h2>Additional data</h2>
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<p>View the <a href="https://static.igem.org/mediawiki/2014/1/1b/Uo2014-raw.pdf" target="_blank">raw flow cytometry data</a>.</p>
             </div>
             </div>
             <div class="pane" id="pane-bricks" hidden>
             <div class="pane" id="pane-bricks" hidden>

Latest revision as of 03:58, 18 October 2014

The Project

Engineering Fate: Cellular decision making and the Tri-Stable switch

Throughout our lives, individual cells make vital decisions that directly affect us. From deciding how to develop, when stem cells differentiation into various cell types, to when to die, as mutated or erroneous cells undergo apoptosis.

We decided to examine how cells make those decisions.

It was hypothesized that a unique tri-stable switch controlled stem cell differentiation, with the three states being an arbitrary state A, B and AB, where both states coexists stably (AB).

Design adapted from Sui Huang, 2009.

For instance, if state A produced blue marbles and B red marbles, the three states would look like this:

Now instead of marbles, lets image A and B as cell types like liver cells and heart cells, and the AB state the undifferentiated state! This of course is a massive over simplification, as A and B in nature are likely transcription factors. Yet, it helps to visualize this switch as such.

(TOP) Human Blastocyst from early development. (LEFT) Human liver cells. (RIGHT) Human heart cells.

This is a primary example of cellular decision making. The 2014 uOttwa iGEM team chose to build this decision making pathway. To do so we created a novel form of gene regulation using activators as repressors.

Potential Applications

Why build such a system? Understanding how this genetic network works and being able to model its behaviour may shed light on how exactly stem cells differentiate. More importantly, it will allow us to engineer cells that implement this synthetic decision-making pathway, and use it in an application such as logic gates.

Alternatively, we may use this system as a unique cellular detector. If A and B are reporters driven by promoters that are sensitive to small molecules such as phosphorous and nitrogen, these cells can monitor the balance between those two. The balance between those two is an important indicator of human pollution, which is indicated by high levels of phosphorous. If one spikes higher than the other, the cell will enter either the A or B state, which would be indicated by their respective reporters. If both spike, it will remain in the AB and indicate an equilibrium.