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| − | {{BostonU_we_tryin}}
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| − | <html>
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| − | <head>
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| − | <style>
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| − | #sideMenu,https://2016.igem.org/ #top_title {display:none;}
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| − | body {background-color:white; }
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| − | #content { padding:0px; width:100%; margin-top:-7px; margin-left:0px;}
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| − | #bodyContent h1, #bodyContent h2, #bodyContent h3, #bodyContent h4, #bodyContent h5 { margin-bottom: 0px; }
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| − | </style>
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| − | </head>
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| − | <div id = "buttononeback">
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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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| − | <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>
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| − | </div>
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| − | <body>
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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;">Phase 1</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;">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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| − | <div id="hover-contentone">
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| − | <br>
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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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| − | <center><img src = "https://2016.igem.org/File:T--BostonU--initialcirc.png" style = "width:80%"></center>
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| − | <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. 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><img style = "width:80%;" src = "https://static.igem.org/mediawiki/2016/3/3f/T--BostonU--ThreeGenes.jpg"></center>
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| − | <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. 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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| − | <center><img src = "https://static.igem.org/mediawiki/2016/f/f6/T--BostonU--Orthogonality.jpg" style = "width:50%;"></center>
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| − | </div>
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| − | <div id="hover-contenttwo">
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| − | <br>
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| − | <p style = "font-size:260%; color:#0071A7; text-align:center;">Phase 2 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;">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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| − | <br>
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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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| − | <br>
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| − | <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><img src = "" style = "width:50%;"></center>
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| − | </div>
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| − | <div id="hover-contentthree">
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| − | <br>
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| − | <p style = "font-size:260%; color:#0071A7; text-align:center;">Phase 2 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;">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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| − |
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| − | <br>
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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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| − |
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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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| − | <br>
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| − | <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><img src = "" style = "width:50%;"></center>
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