Difference between revisions of "Team:BostonU/HomeOne"

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<br>
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Project Design
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</div>
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<div id = "buttonback">
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#buttonnametwotwo {
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<div style = "margin:3.75vh 0vh 0vh 0vh;">
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<a href="https://2016.igem.org/Team:BostonU/Design" style = "text-align:center; font-size:200%; color:#0071A7;"><</a>
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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;">Phase 1</p>
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}
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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;">Phase 2</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;">Phase 3</p>
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</div>
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</div>
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<br>
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#twit, #face, #snap, #email {
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<p style = "font-size:260%; color:#0071A7; text-align:center;">Phase 1 Results</p>
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<center><hr style= "width:550px; height: 4px; background-color:#0071A7"></center><br>
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<p style = "text-indent:70px; font-size:150%; padding:25px 150px 50px 150px; color:#0071A7;">The goal of phase 1 to establish a consistent method of gene activation through dCAS9-VPR and well characterize the components of our system. Using MIT's CRISPR  optimization tool, we generated 20 sequences to be used as our gRNA and target site. The dCAS9-VPR complex, the target sequence and reporter gene, and the gRNA expression vectors were constructed and transfected into HEK293 cells. Another set of transfections took place simultaneously with the same materials minus the gRNA expression vector as a negative control. The fold increase between the basal level of expression from the control and the activated level of expression was then recorded. The results can be found below.</p>
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}
  
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<center><img src = "https://static.igem.org/mediawiki/2016/a/aa/T--BostonU--InitialData.jpg" style = "width:80%"></center>
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#twit:hover, #face:hover, #snap:hover, #email:hover {
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transform:scale(1.5)
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}
  
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</style>
  
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<body>
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<div id = "container">
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<div id = "one" class = "show">
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<img style = "width:100%;" src = "https://static.igem.org/mediawiki/2016/3/35/T--BostonU--Geminisymbol.jpg" id = "gem">
 +
</div>
  
 +
<div style = "width:80%;" id = "two" class = "show">
 
<br>
 
<br>
−
<p style = "text-indent:70px; font-size:150%; padding:25px 150px 50px 150px; color:#0071A7;">The goal of phase 1 to establish a consistent method of gene activation through dCAS9-VPR. Using MIT's CRISPR  optimization tool, we generated 20 sequences to be used as our gRNA and target site. The dCAS9-VPR complex, the target sequence and reporter gene, and the gRNA expression vectors were constructed and transfected into HEK293 cells. Another set of transfections took place simultaneously with the same materials minus the gRNA expression vector as a negative control. The fold increase between the basal level of expression from the control and the activated level of expression was then recorded. The results can be found below. Here are some extra words to make it seem like this is a different paragraph than the rest when really, it's not. It's just the same thing. But I want to make it look different, so there you go. Let's just add a few more words, and there we go. That should do it.</p>
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<center style = "font-size:280%; font-family:Trebuchet MS; color:#0071A7;">Our Project</center>
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<br><center><hr style= "width:702px; height: 3px; background-color:#0071A7"></center>
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<center><img style = "width:80%;" src = "https://static.igem.org/mediawiki/2016/3/3f/T--BostonU--ThreeGenes.jpg"></center>
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<center><p style = "width:80%; color:#0071A7; font-size:125%; padding:15px;">In nature, some cells have the ability to detect if certain environmental signals such as hormones, drugs, or wavelengths of light are present or absent. These digital signals can then determine the analog expression level of certain genes. Our project aims to replicate this natural process through a dCas9 based system. Our system, Gemini, is able to recognize the presence (represented by a 1) or the absence (represented by a 0) of several environmental signals and change a gene's level of expression based on what combination of signals it is registering. For a more detailed description, start the Project Tour.</p>
 +
<img style = "width:28%; padding:0px 5%;" src = "https://static.igem.org/mediawiki/2016/5/51/T--BostonU--Signals.png">
 +
<img style = "width:25%; padding:0px 5%;" src = "https://static.igem.org/mediawiki/2016/6/6a/T--BostonU--digi.png">
 +
</center>
 +
<div id = "buttonname">
 
<br>
 
<br>
−
<p style = "text-indent:70px; font-size:150%; padding:25px 150px 50px 150px; color:#0071A7;">The goal of phase 1 to establish a consistent method of gene activation through dCAS9-VPR. Using MIT's CRISPR  optimization tool, we generated 20 sequences to be used as our gRNA and target site. The dCAS9-VPR complex, the target sequence and reporter gene, and the gRNA expression vectors were constructed and transfected into HEK293 cells. Another set of transfections took place simultaneously with the same materials minus the gRNA expression vector as a negative control. The fold increase between the basal level of expression from the control and the activated level of expression was then recorded. The results can be found below.</p>
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Project Tour
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</div>
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<center><img src = "https://static.igem.org/mediawiki/2016/f/f6/T--BostonU--Orthogonality.jpg" style = "width:50%;"></center>
+
  
 +
<div id = "button">
 +
<a href="https://2016.igem.org/Team:BostonU/Description"><img style = "width:5vw" src = "https://static.igem.org/mediawiki/2016/f/ff/T--BostonU--bluefor.png"></a>
 +
</div>
 +
</div>
 
</div>
 
</div>
  
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<div style = "width:80%;" id = "three" class = "show">
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<br>
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<center style = "font-size:280%; font-family:Trebuchet MS; color:#bd162a;">HP Projects</center>
 +
<br><center><hr style= "width:702px; height: 3px; background-color:#bd162a;"></center>
 +
<center><p style = "width:80%; color:#bd162a; font-size:125%; padding:15px;">In addition to creating Gemini, our team also aimed to go beyond the lab space and contribute to the synthetic biology community in other ways. In addition to volunteering for several outreach programs, we also developed two human practices projects. The first was creating a set of interactive discussion forums meant to make synthetic biology more accessible to the public. The second was a blog intended to inform synthetic biologists about important considerations and implications in patent law and intellectual property. To see a timeline of how we created our forums or explore our blog posts, click the buttons below.</p>
 +
<img style = "float:left; width:30%; padding:2% 0% 2% 10%;" src = "https://static.igem.org/mediawiki/2016/e/e0/T--BostonU--Summer_Pathways.jpg">
 +
<img style = "width:30%; padding:2%; float:left;" src = "https://static.igem.org/mediawiki/2016/f/f8/T--BostonU--Forums.jpg">
 +
</center>
  
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<div id="hover-contenttwo">
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<div id = "buttonnametwo">
 
<br>
 
<br>
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<p style = "font-size:260%; color:#0071A7; text-align:center;">Phase 2 Results</p>
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Forums
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<center><hr style= "width:550px; height: 4px; background-color:#0071A7"></center><br>
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</div>
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<p style = "text-indent:70px; font-size:150%; padding:25px 150px 50px 150px; color:#0071A7;">With our parts of our project well characterized, we began experimenting with achieving a variety of consistent expression levels with our dCAS9-VPR activator. Our first strategy was to attempt multimerizing the target operators, placing two and then three operators up stream of the gene of interest using two of our operators. During this experiment, we also tested the effect of spacing the target operators with a different number of intervening base pairs. We tested with 0, 3, 6, 12, and 24 intervening base pairs. The results of these experiments yielded a predictable, linear correlation between the number of operators and the expression level and the number of base pairs in the intervening sequences. The results can be found below: </p>
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<center><img src = "https://static.igem.org/mediawiki/2016/2/27/T--BostonU--multi.png" style = "width:80%"></center>
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<div id = "buttontwotwo">
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<a href="https://2016.igem.org/Team:BostonU/HP/Silver"><img style = "width:5vw" src = "https://static.igem.org/mediawiki/2016/1/1d/T--BostonU--redfor.png"></a>
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</div>
  
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<div id = "buttonnametwotwo">
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<br>
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Blog
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</div>
  
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<div id = "buttontwo">
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<a href="https://2016.igem.org/Team:BostonU/HP/Gold"><img style = "width:5vw" src = "https://static.igem.org/mediawiki/2016/1/1d/T--BostonU--redfor.png"></a>
 +
</div>
  
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<br>
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</div>
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<p style = "text-indent:70px; font-size:150%; padding:25px 150px 50px 150px; color:#0071A7;">There were some anomalies within this data set. The target operators with 0 and 12 base pair spacing had lower expression than expected. One explanation is that when the operators are at 0 and 12 base pairs apart, they are on the same side of the DNA helix and crowd each other out. Once we eliminated these results, were able to create a smooth progression in expression level across several levels of expression, as seen below.</p>
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<center><img style = "width:80%;" src = "https://static.igem.org/mediawiki/2016/f/fb/T--BostonU--chosen.png"></center>
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<div style = "width:80%;" id = "four" class = "show">
 
<br>
 
<br>
−
<p style = "text-indent:70px; font-size:150%; padding:25px 150px 50px 150px; color:#0071A7;">Finally, we realized that while we could achieve a wide range of high expression levels, it is sometimes favorable to lower expression levels below the standard single operator expression level. In order to accomplish this, we mutated our operators, replacing a base pair at a time. We tested 20 versions of 2 of our operator plasmids. Each version had one of the twenty base pairs in the twenty base pair operator mutated, so that there was a single mismatch between the operator and the guide RNA attached to the dCAS9-VPR system. By changing the location of the single mismatch, we were able to create another smooth curve of changing expression levels, this time lowering expression levels. The expression levels can be found in the graph below.</p>
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<center style = "font-size:230%; font-family:Trebuchet MS; color:#507f54;">For the Judges</center>
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<br><center><hr style= "width:702px; height: 3px; background-color:#507f54;"></center>
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<center><img src = "" style = "width:50%;"></center>
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<center><img src = "https://static.igem.org/mediawiki/2016/9/9e/T--BostonU--Medals.png" style = "width:60%;"></center>
 +
<p style = "text-align:center; color:#507f54; font-size:175%;">A full list of how we met each medal requirement can be found here:</p></center>
  
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<div id = "buttonthree">
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<a href="https://2016.igem.org/Team:BostonU/Medal_Criteria"><img style = "width:6vw" src = "https://static.igem.org/mediawiki/2016/c/c4/T--BostonU--Greenforward.png"></a>
 +
</div>
 
</div>
 
</div>
  
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<div id="hover-contentthree">
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<div style = "width:80%;" id = "five" class = "show">
 
<br>
 
<br>
−
<p style = "font-size:260%; color:#0071A7; text-align:center;">Phase 3 Results</p>
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<center style = "font-size:280%; font-family:Trebuchet MS; color:#0071A7;">Team Gemini</center>
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<center><hr style= "width:550px; height: 4px; background-color:#0071A7"></center><br>
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<div style = "width:30px; height:30px; background-color:red; z-index:200; position:relative; left:19.5%; top:150px;"></div>
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<p style = "text-indent:70px; font-size:150%; padding:25px 150px 50px 150px; color:#0071A7;">Now that we could achieve a variety of expression levels, we began integrating our system into recombination based circuitry. The circuit we used could release a different gRNA for every combination of drugs introduced. Before integrating our multimerized plasmids, we first tested the circuit by making each gRNA correspond to a different fluorescent reporter. The data for this experiment can be found below. There were four possible combinations of two drugs, and each combination yielded a relatively high expression of its corresponding fluorescent gene.</p>
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<div style = "width:30px; height:30px; background-color:red; z-index:200; position:relative; left:53%; top:270px;"></div>
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<div style = "width:30px; height:30px; background-color:red; z-index:200; position:relative; left:80%; top:310px;"></div>
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<br><center><hr style= "width:702px; height: 3px; background-color:#0071A7;"></center>
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<center><img src = "https://static.igem.org/mediawiki/2016/5/5c/T--BostonU--Team_Collage.png" style = "width:80%"><center>
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<div id = "buttonname">
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<br>
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Attributions
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</div>
  
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<center><img src = "https://static.igem.org/mediawiki/2016/2/21/T--BostonU--initialcirc.png" style = "width:80%"></center>
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<div id = "button">
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<a href="https://2016.igem.org/Team:BostonU/Attributions"><img style = "width:5vw" src = "https://static.igem.org/mediawiki/2016/f/ff/T--BostonU--bluefor.png"></a>
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</div>
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</div>
  
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</div>
  
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<div style = "width:10%; background-color:white; color:#0071A7; text-align:center; font-size:300%; position:absolute; z-index:100; left: 90%; cursor:pointer;" id = "forward"><div style = "height:300px;"></div>><div style = "height:300px;"></div></div>
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<div style = "width:10%; cursor:pointer; height:600px; background-color:white; color:#0071A7; text-align:center; font-size:300%; position:absolute; left:-1%; z-index:100;" id = "reverse"><div style = "height:300px;"></div><<div style = "height:300px;"></div></div>
  
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<div style = "width:80%; height:600px;" id = "six" class = "show">
 
<br>
 
<br>
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<p style = "text-indent:70px; font-size:150%; padding:25px 150px 50px 150px; color:#0071A7;">Results</p>
 
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<center><img style = "width:80%;" src = ""></center>
 
 
<br>
 
<br>
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<p style = "text-indent:70px; font-size:150%; padding:25px 150px 50px 150px; color:#0071A7;">Results</p>
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<center style = "font-size:280%; font-family:Trebuchet MS; color:#507f54;">Thank You to Our Sponsors</center>
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<br><center><hr style= "width:702px; height: 3px; background-color:#507f54;"></center>
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<center><img src = "" style = "width:50%;"></center>
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<center><img src = "https://static.igem.org/mediawiki/2016/e/eb/T--BostonU--Sponsors.jpg" style = "width:80%"><center>
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</div>
  
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<div style = "width:100%; height:600px;"></div>
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<br><br><br>
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<div id = "media">
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<center style = "font-size:230%; padding:25px; color:#0071A7;">Follow us on:</center>
 
</div>
 
</div>
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<br><br><br><br><br><br><br><br>
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<center>
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</div>
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<a href = "https://twitter.com/buigemwl2016"> <img id = "twit" src = "https://static.igem.org/mediawiki/2016/e/ee/T--BostonU--twitterlogo.png"> </a>
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<a href = "https://mail.google.com/mail/?view=cm&fs=1&to=bu.igemwl2016@gmail.com&su=&body=&bcc="><img id = "email" src = "https://static.igem.org/mediawiki/2016/c/c3/T--BostonU--gmaillogo.png"></a>
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<a href = "https://www.facebook.com/buigemwl2016"> <img id = "face" src = "https://static.igem.org/mediawiki/2016/b/b7/T--BostonU--facebooklogo.jpg"></a>
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<img id =  "snap" src = "https://static.igem.org/mediawiki/2016/8/8d/T--BostonU--snapchatlogo.png">
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<br><br><br><br><br><br><br>
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Revision as of 15:11, 8 October 2016


Our Project

In nature, some cells have the ability to detect if certain environmental signals such as hormones, drugs, or wavelengths of light are present or absent. These digital signals can then determine the analog expression level of certain genes. Our project aims to replicate this natural process through a dCas9 based system. Our system, Gemini, is able to recognize the presence (represented by a 1) or the absence (represented by a 0) of several environmental signals and change a gene's level of expression based on what combination of signals it is registering. For a more detailed description, start the Project Tour.


Project Tour

HP Projects

In addition to creating Gemini, our team also aimed to go beyond the lab space and contribute to the synthetic biology community in other ways. In addition to volunteering for several outreach programs, we also developed two human practices projects. The first was creating a set of interactive discussion forums meant to make synthetic biology more accessible to the public. The second was a blog intended to inform synthetic biologists about important considerations and implications in patent law and intellectual property. To see a timeline of how we created our forums or explore our blog posts, click the buttons below.


Forums

Blog

For the Judges

A full list of how we met each medal requirement can be found here:


Team Gemini


Attributions
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Thank You to Our Sponsors




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