Difference between revisions of "Team:BostonU/Design"

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2016.igem.org/Judging/Awards#Special_Prizes"> design special prize</a>. </p>
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
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<a href="https://2016.igem.org/Team:BostonU/Abstract_and_Motivation"><img style = "width:5vw" src = "https://static.igem.org/mediawiki/2016/a/a8/T--BostonU--blueback.png"></a>
 
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<br>
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Motivation
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<div id = "buttonone" style = "font-size:175%;">
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<br>
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Proof of Concept
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<a href="https://2016.igem.org/Team:BostonU/Proof"><img style = "width:5vw" src = "https://static.igem.org/mediawiki/2016/f/ff/T--BostonU--bluefor.png"></a>
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<p>
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By talking about your design work on this page, there is one medal criterion that you can attempt to meet, and one award that you can apply for. If your team is going for a gold medal by building a functional prototype, you should tell us what you did on this page.
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<div style = "width:80%; margin:0 auto;">
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<div id = "design" style = "font-size:300%; padding:75px 50px 3px 50px; text-align:center; color:#0071A7;">Design</div>
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<br><center><hr style= "width:60%; height: 3px; background-color:#0071A7"></center><br>
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<p style = "font-size:150%; padding:5px 150px 5px 150px; color:#0071A7;">Seeing that the problem we wanted to address had a natural duality to it (digital and analog), we decided to develop Gemini. This system's name refers to the twins Castor and Pollux in Greek mythology. What attracted us most to this name was the fact that while each of the twins was a hero in their own regard, they were far more powerful together. While digital and analog promoter networks are individually impressive, the ability to control both networks through one system is the ultimate goal. To obtain our system of well characterized mammalian digital and analog promoters, we divided our research into three technical aims:</p>
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<div style = "width:60%; position:relative; left:20%;">
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<ul>
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<li style = "font-size:150%; color:#0071A7; padding: 10px 0px;">Development of a digital promoter library</li>
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<li style = "font-size:150%; color:#0071A7; padding: 10px 0px;">Expansion into an analog promoter library</li>
 +
<li style = "font-size:150%; color:#0071A7; padding: 10px 0px;">Integration of our parts into genetic circuit contexts</li>
 +
</ul>
 +
</div>
 +
 
 +
<p style = "font-size:150%; padding:5px 150px 20px 150px; color:#0071A7;"> To complete these goals we made use of three classes of plasmids: a constitutive guide RNA expressing vector, a guide RNA operator reporter vector driven by a minimal CMV, and a constitutive dCas9-VPR complex. dCas9, like its sister system Cas9, works by using CRISPR’s targeting system and a guide RNA to find a specific sequence. Where they differ is in how the proteins interact with the genome. While Cas9 has a nuclease that can cleave the DNA, dCas9’s nuclease is catalytically inhibited. This results in a protein that can be localized to a region of DNA but cannot physically modify it. When fused, however, to the VPR, a strong activating complex of transcription regulators, the dCas9 becomes an activating complex that can drive the production of a gene of interest. </p>
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 +
<center><img src = "https://static.igem.org/mediawiki/2016/3/37/T--BostonU--ProjectDescription_dCas9_explanation.png" style = "padding:0px 0px 50px 0px; width:50%;"></center>
 +
 
 +
<p style = "font-size:150%; padding:5px 150px 20px 150px; color:#0071A7;"> In our work, those genes of interest were fluorescent proteins. The benefit of using fluorescent proteins is that they are easily assayable. The two assaying techniques used throughout our research were flow cytometry and fluorescent microscopy (the latter courtesy of Worcester Polytechnic Institute’s iGEM Team).  
 
</p>
 
</p>
  
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<br><center><hr style= "width:60%; height: 3px; background-color:#0071A7"></center><br>
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<br>
  
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<p>This is a prize for the team that has developed a synthetic biology product to solve a real world problem in the most elegant way. The students will have considered how well the product addresses the problem versus other potential solutions, how the product integrates or disrupts other products and processes, and how its lifecycle can more broadly impact our lives and environments in positive and negative ways.</p>
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<div id = "container">
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<center>
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<div class = "thebuttons" id = "parentone">
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<p style = "text-align:center; font-size:200%; padding:0px 0px 0px 0px;">Digital</p>
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</div>
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<div class = "thebuttons" id = "parenttwo" style="margin:0vw 12vw 0vw 12vw;">
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<p style = "text-align:center; font-size:200%; padding:0px 0px 0px 0px;">Analog</p>
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</div>
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<div class = "thebuttons" id = "parentthree">
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<p style = "text-align:center; font-size:200%; padding:0px 0px 0px 0px;">Circuits</p>
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</div>
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</div>
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</center>
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<br>
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<center><div style= "color:#bd162a; font-size:150%; width:100%;">Click on each button to read how we designed our system to meet each aim.</div></center><br>
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<div>
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<div id = "contone" class = "cont">
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<br><br><center style = "font-size:225%; color:#0071A7;">Aim 1:</center><br>
 +
<center style = "font-size:200%; color:#0071A7;">Developing a Digital Library</center>
 +
 
 +
<p style = "font-size:150%; padding:25px 150px 20px 150px; color:#0071A7;">We began our work by developing the digital promoter library. Before moving into experimentation, we made several design considerations. First, our system had to be orthogonal to the human genome. We did not want to activate off target genes in the human genome. Although not immediate, we envision our work having long reaching applications in therapeutics and immunotherapy, and thus we wanted to begin optimizing our system accordingly. </p>
 +
 
 +
 
 +
<p style = "font-size:150%; padding:5px 150px 20px 150px; color:#0071A7;">Our next design goal was to demonstrate low basal activity when necessary components were absent, and high expression activity when all components were present. This behavior needed to be maintained across multiple genes of interest.</p>
 +
 
 +
<p style = "font-size:150%; padding:5px 150px 20px 150px; color:#0071A7;">Finally, we needed to motivate that components of our system were mutually orthogonal. We wanted to make sure that there was no cross talk that would generate undesired outputs under the wrong conditions.</p>
 +
 
 +
 
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<center><img src = "https://static.igem.org/mediawiki/2016/3/38/T--BostonU--SingleOp-DesignPage.png" style = "padding:0px 0px 50px 0px;; width:50%;"></center>
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</div>
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</div>
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<div>
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<div id = "conttwo" class = "cont">
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<br><br><br><center style = "font-size:225%; color:#0071A7;">Aim 2:</center><br>
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<center style = "font-size:200%; color:#0071A7;">Expanding to an Analog Library</center>
 +
 
 +
<p style = "font-size:150%; padding:25px 150px 20px 150px; color:#0071A7;">After establishing the digital library, we moved to expand into our full analog library. We did this using two main hypotheses: the multimerization of binding sites would increase expression and the mutation of binding sites would decrease expression. In the case of the former, the multimerization would recruit a greater number of dCas9-VPR’s upstream of the minimal promoter. In the case of the former, mutations should decrease the binding affinity of the dCas9-VPR. In addition to those parameters, we also increased the distance between binding sites on the multimerized plasmids in another attempt to increase expression.
 +
Once the library was expanded out into the analog domain, we begun integrating our library into genetic logic circuits. The motivation behind doing this was is once again two fold (catching a theme here… wink… wink...Gemini). First, genetic circuits represent one of synthetic biology’s most powerful tools. Circuits have become integral in our search for better immunotherapy procedures. Also, genetic circuits grant a scientist a greater capacity to mimic naturally cellular behavior for synthetic operations. </p>
  
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<p>
 
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If you are working on art and design as your main project, please join the art and design track. If you are integrating art and design into the core of your main project, please apply for the award by completing this page.
 
 
</p>
 
</p>
  
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<center><img src = "https://static.igem.org/mediawiki/2016/d/d9/T--BostonU--multimerization.png" style = "padding:0px 0px 50px 0px;; width:80%;"></center>
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</div>
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</div>
  
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<p>Teams who want to focus on art and design should be in the art and design special track. If you want to have a sub-project in this area, you should compete for this award.</p>
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<div>
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<div id = "contthree" class = "cont">
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<br><br><br><center style = "font-size:225%; color:#0071A7;">Aim 3:</center><br>
 +
<center style = "font-size:200%; color:#0071A7;">Integrating our Library into Genetic Circuit Contexts</center>
 +
 
 +
<p style = "font-size:150%; padding:25px 150px 20px 150px; color:#0071A7;">There were two distinct class of circuits we aimed to develop. The first is a combinatorial digital logic circuit. These circuits are defined by their binary outputs as well as their ignorance towards input order. In a biological context, a binary output would be the expression or absence of expression of a gene of interest. The second type of circuit is the combinatorial analog logic circuit. These circuit are defined by their analog output of a single gene as well as their ignorance towards input order. In a biological context, this behavior can be modelled by increasing the expression of gene as one shifts from a given logical state to another logical state.
 +
</p>
 +
 
 +
<p style = "font-size:150%; padding:5px 150px 20px 150px; color:#0071A7;">Each design parameter mentioned above required its own distinct set of experiments to prove our system could achieve them. Ultimately, we were able to prove that Gemini could fulfill all design parameters. The evidence of this can be seen under our Proof of Concept.
 +
</p>
 +
 
 +
<center><img src = "https://static.igem.org/mediawiki/2016/0/06/T--BostonU--RealRecombinase.png" style = "padding:0px 0px 50px 0px;; width:80%;"></center>
 
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Latest revision as of 21:53, 19 October 2016


Motivation

Proof of Concept
Design


Seeing that the problem we wanted to address had a natural duality to it (digital and analog), we decided to develop Gemini. This system's name refers to the twins Castor and Pollux in Greek mythology. What attracted us most to this name was the fact that while each of the twins was a hero in their own regard, they were far more powerful together. While digital and analog promoter networks are individually impressive, the ability to control both networks through one system is the ultimate goal. To obtain our system of well characterized mammalian digital and analog promoters, we divided our research into three technical aims:

  • Development of a digital promoter library
  • Expansion into an analog promoter library
  • Integration of our parts into genetic circuit contexts

To complete these goals we made use of three classes of plasmids: a constitutive guide RNA expressing vector, a guide RNA operator reporter vector driven by a minimal CMV, and a constitutive dCas9-VPR complex. dCas9, like its sister system Cas9, works by using CRISPR’s targeting system and a guide RNA to find a specific sequence. Where they differ is in how the proteins interact with the genome. While Cas9 has a nuclease that can cleave the DNA, dCas9’s nuclease is catalytically inhibited. This results in a protein that can be localized to a region of DNA but cannot physically modify it. When fused, however, to the VPR, a strong activating complex of transcription regulators, the dCas9 becomes an activating complex that can drive the production of a gene of interest.

In our work, those genes of interest were fluorescent proteins. The benefit of using fluorescent proteins is that they are easily assayable. The two assaying techniques used throughout our research were flow cytometry and fluorescent microscopy (the latter courtesy of Worcester Polytechnic Institute’s iGEM Team).




Digital

Analog

Circuits


Click on each button to read how we designed our system to meet each aim.



Aim 1:

Developing a Digital Library

We began our work by developing the digital promoter library. Before moving into experimentation, we made several design considerations. First, our system had to be orthogonal to the human genome. We did not want to activate off target genes in the human genome. Although not immediate, we envision our work having long reaching applications in therapeutics and immunotherapy, and thus we wanted to begin optimizing our system accordingly.

Our next design goal was to demonstrate low basal activity when necessary components were absent, and high expression activity when all components were present. This behavior needed to be maintained across multiple genes of interest.

Finally, we needed to motivate that components of our system were mutually orthogonal. We wanted to make sure that there was no cross talk that would generate undesired outputs under the wrong conditions.




Aim 2:

Expanding to an Analog Library

After establishing the digital library, we moved to expand into our full analog library. We did this using two main hypotheses: the multimerization of binding sites would increase expression and the mutation of binding sites would decrease expression. In the case of the former, the multimerization would recruit a greater number of dCas9-VPR’s upstream of the minimal promoter. In the case of the former, mutations should decrease the binding affinity of the dCas9-VPR. In addition to those parameters, we also increased the distance between binding sites on the multimerized plasmids in another attempt to increase expression. Once the library was expanded out into the analog domain, we begun integrating our library into genetic logic circuits. The motivation behind doing this was is once again two fold (catching a theme here… wink… wink...Gemini). First, genetic circuits represent one of synthetic biology’s most powerful tools. Circuits have become integral in our search for better immunotherapy procedures. Also, genetic circuits grant a scientist a greater capacity to mimic naturally cellular behavior for synthetic operations.




Aim 3:

Integrating our Library into Genetic Circuit Contexts

There were two distinct class of circuits we aimed to develop. The first is a combinatorial digital logic circuit. These circuits are defined by their binary outputs as well as their ignorance towards input order. In a biological context, a binary output would be the expression or absence of expression of a gene of interest. The second type of circuit is the combinatorial analog logic circuit. These circuit are defined by their analog output of a single gene as well as their ignorance towards input order. In a biological context, this behavior can be modelled by increasing the expression of gene as one shifts from a given logical state to another logical state.

Each design parameter mentioned above required its own distinct set of experiments to prove our system could achieve them. Ultimately, we were able to prove that Gemini could fulfill all design parameters. The evidence of this can be seen under our Proof of Concept.