Difference between revisions of "Team:BostonU/Design"

 
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Experimental Results
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<a href="https://2016.igem.org/Team:BostonU/HomeOne"><img style = "width:5vw" src = "https://static.igem.org/mediawiki/2016/a/a8/T--BostonU--blueback.png"></a>
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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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Description
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Motivation
 
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Proof of Concept
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<a href="https://2016.igem.org/Team:BostonU/Design"><img style = "width:5vw" src = "https://static.igem.org/mediawiki/2016/f/ff/T--BostonU--bluefor.png"></a>
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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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<div id = "design" style = "font-size:300%; padding:75px 50px 25px 50px; text-align:center; color:#0071A7;">Design</div>
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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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<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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<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>
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<li style = "font-size:150%; color:#0071A7; padding: 10px 0px;">Integration of our parts into genetic circuit contexts</li>
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<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>
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<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).
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<br><br><center style = "font-size:225%; color:#0071A7;">Phase 1:</center><br>
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<center style = "font-size:200%; color:#0071A7;">Gene Activation Component</center>
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<p style = "text-align:center; font-size:200%; padding:0px 0px 0px 0px;">Digital</p>
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<p style = "text-align:center; font-size:200%; padding:0px 0px 0px 0px;">Analog</p>
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<p style = "text-align:center; font-size:200%; padding:0px 0px 0px 0px;">Circuits</p>
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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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<p style = "font-size:150%; padding:25px 150px 20px 150px; color:#0071A7;">In order to make genes activate in response to certain signals, our system first needed a method to activate genes in general. We chose CRISPR/dCAS9-VPR as an activator. We chose  dCAS9 due to its ease of use and its ability to target specific DNA sequences. dCAS9-VPR targets specific sequences by binding to specialized RNA. Part of this RNA (gRNA) contains 20 base pairs that will act as a guide, guiding the dCAS9 to the complimentary 20 base pairs found upstream of a gene one wishes target. This can be seen in the info-graphic below:</p>
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<br><br><center style = "font-size:225%; color:#0071A7;">Aim 1:</center><br>
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<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>
  
−
<center><img src = "https://static.igem.org/mediawiki/2016/3/37/T--BostonU--ProjectDescription_dCas9_explanation.png" style = "padding:0px 0px 50px 0px; width:80%;"></center>
 
  
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<br><br><br><center style = "font-size:225%; color:#0071A7;">Phase 2:</center><br>
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<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>
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<center style = "font-size:200%; color:#0071A7;">Analog Expression System</center>
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<p style = "font-size:150%; padding:25px 150px 20px 150px; color:#0071A7;">Once we were able to activate genes, we then expanded our system to activate genes to different levels, thereby achieving the graded analog expression level that we desired from our system. To accomplish this, we multermerized the 20 base pair target sequence, placing multiple copies
+
<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>
−
of the target sequence upstream of the gene. By varying the number of copies, we were able to create a gradient of expression. The more target sequences we added, the more the gene was activated. This is illustrated in the image below:</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 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>
 +
 
 +
</p>
  
 
<center><img src = "https://static.igem.org/mediawiki/2016/d/d9/T--BostonU--multimerization.png" style = "padding:0px 0px 50px 0px;; width:80%;"></center>
 
<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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<br><br><br><center style = "font-size:225%; color:#0071A7;">Phase 3:</center><br>
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<center style = "font-size:200%; color:#0071A7;">Signal Integration Components</center>
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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>
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<center style = "font-size:200%; color:#0071A7;">Integrating our Library into Genetic Circuit Contexts</center>
  
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<p style = "font-size:150%; padding:25px 150px 20px 150px; color:#0071A7;">Finally, once we completed phase one and two, we expanded our system once again. We utilized the recombinase system BLADE. BLADE allows for digital activation of different genes by using inducible recombinase proteins to excise DNA. When different combinations of recombinases are activated by digital signals, different combinations of DNA are excised. Based on what parts of the circuit are excised decides which one of several gRNA's to release. Once a gRNA is released, it will bind to a dCAS9-VPR and guide the activator to the gene with the corresponding operator. When a new combination of recombinases are activated, a different gRNA is released, guiding the activator to a different gene. The results of BLADE can be seen in the graph below.  
+
<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.  
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<br>
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</p>
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<img src = "" style = "width:20%; height:100px;">  
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<br>
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<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.
−
As seen above, this is similar to our system. Except instead of activating different genes, we want to integrate the same gene to different levels based on the signal input. We adapted BLADE by integrating our well characterized parts into its framework. Instead of each gRNA in the circuit corresponding to the operator upstream of a different gene, all the unique gRNA's would correspond to one gene. The difference is that each gRNA targets a different copy of the gene. Each copy has a different number of target sequences for the a specific gRNA sequence to bind to. A diagram of this process can be found below. BLADE also allows us to integrate more signals and more recognition site, achieving a truth table with up to three inputs. The finished product relied on digital activation, (the recombinases are activated by the digital prescience or absence of a signal such as a hormone) and gave rise to different levels of analog gene expression, as stated in our goal.</p>
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<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.