Difference between revisions of "Team:Alverno CA/Design"

 
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<title>Alverno iGEM 2016</title>
 
<title>Alverno iGEM 2016</title>
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  <link rel="icon" href="https://static.igem.org/mediawiki/2016/thumb/5/58/T--Alverno_CA--Alverno_iGEM_2016_Logo.png/600px-T--Alverno_CA--Alverno_iGEM_2016_Logo.png">
 
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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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<h2><center>Design</center></h2>
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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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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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<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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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.
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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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<h5>The goal of of our project is to counteract a problem in synthetic biology known as supercoiling. Supercoiling is a possible consequence that can occur in the construction of a multi-genetic circuit. When the two genes transcribe, the double helix is opened in order to allow for the expression for the genes, however, the dna in between the two genes receive extraordinary amounts of tension when both genes try to transcribe at the same time, which can lead to the over transcription of one gene, and the under expression of the other gene. This can greatly affect the predictability of gene expression.  </h5>
  
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<h5> To test if we could reduce the supercoiling between these two genes, we proposed several different strategies that we felt may alleviate the problem. The first method we tried was adding random base pair sequences in between the two genes, with lengths of 500bp or 1000bp. We created this in the hope that the spacer in between the two genes would alleviate the tension that creates the supercoiling. Another one of our methods was to use a dCas9 clamp between the RFP and GFP to clamp down between the two genes and stop the supercoiling.
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In order to see whether or not the problem of supercoiling was reduced, we had to first construct the plasmid circuits used for our experiment by using Golden Gate Assembly. First, we designed primers to amplify our parts off current iGEM parts using the online DNA design editor, <a href="https://benchling.com/sclamons/f_/fBGWWXin-parts/?sort=name"> Benchling.</a>For our experiment, we have used RFP and GFP and parts from the iGEM inventory: <a href="http://parts.igem.org/Part:BBa_J04450"> BBa_J04450</a> and <a href="http://parts.igem.org/Part:BBa_I13522"> BBa_I13522,</a> respectively. These parts were amplified off and ligated together in varying orientations (see picture designs) with a spacer part between them (either 500bp spacer, 1000bp spacer, or a dcas9 clamp site spacer) and finally a vector, or backbone piece, with Chloramphenicol or Kanamycin resistance. <h5>
  
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<img class="icons"src="https://static.igem.org/mediawiki/2016/a/ad/T--Alverno_CA--Katie%27s_drawing_1.png" style=width:200px;>
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<h5>The RFP gene and the GFP gene with transcription occurring in opposite directions away from the dCas 9 binding sites, which separate the two genes.</h5>
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<img class="icons"src="https://static.igem.org/mediawiki/2016/7/7d/T--Alverno_CA--Katie%27s_drawing_2.png" style=width:200px;>
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<h5>The GFP gene being transcribed towards the dCas9 binding sites and the RFP gene, while the RFP gene is being transcribed away from the GFP gene.</h5>
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<img class="icons"src="https://static.igem.org/mediawiki/2016/f/fb/T--Alverno_CA--Katie%27s_drawing_3.png" style=width:200px;>
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<h5>The RFP gene and GFP gene while transcription is occurring towards each other, as well as in the direction of the dCas 9 binding sites.</h5>
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<img class="icons"src="https://static.igem.org/mediawiki/2016/f/f7/T--Alverno_CA--Katie%27s_drawing_4.png" style=width:200px;>
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<h5>The RFP gene being transcribed towards the dCas 9 binding sites and the GFP gene, while the GFP gene is being transcribed away from RFP gene.</h5>
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<img class="icons"src="https://static.igem.org/mediawiki/2016/7/77/T--Alverno_CA--Katie%27s_drawing_6.png" style=width:200px;>
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<h5>The RFP gene and GFP gene are switched. The RFP and the GFP with transcription occurring in opposite directions away from the dCas 9 binding sites, which separate the two genes.</h5>
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<img class="icons"src="https://static.igem.org/mediawiki/2016/0/0c/T--Alverno_CA--Katie%27s_drawing_5.png" style=width:200px;>
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<h5>RFP and GFP are switched. The RFP gene being transcribed towards the dCas9 binding sites and the GFP gene while the GFP gene being transcribed away from the RFP gene.</h5>
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<img class="icons"src="https://static.igem.org/mediawiki/2016/c/cd/T--Alverno_CA--Katie%27s_drawing_7.png" style=width:200px;>
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<h5>RFP and GFP are switched. The RFP gene and GFP gene while transcription is occurring towards each other, as well as in the direction of the dCas 9 binding sites.</h5>
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<img class="icons"src="https://static.igem.org/mediawiki/2016/a/a4/T--Alverno_CA--Katie%27s_drawing_8.png" style=width:200px;>
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<h5>RFP and GFP are switched. The GFP gene being transcribed towards the dCas 9 binding sites and the RFP gene, while the RFP gene is being transcribed away from the GFP gene.</h5>
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Latest revision as of 02:44, 20 October 2016

Alverno iGEM 2016

Design

The goal of of our project is to counteract a problem in synthetic biology known as supercoiling. Supercoiling is a possible consequence that can occur in the construction of a multi-genetic circuit. When the two genes transcribe, the double helix is opened in order to allow for the expression for the genes, however, the dna in between the two genes receive extraordinary amounts of tension when both genes try to transcribe at the same time, which can lead to the over transcription of one gene, and the under expression of the other gene. This can greatly affect the predictability of gene expression.


To test if we could reduce the supercoiling between these two genes, we proposed several different strategies that we felt may alleviate the problem. The first method we tried was adding random base pair sequences in between the two genes, with lengths of 500bp or 1000bp. We created this in the hope that the spacer in between the two genes would alleviate the tension that creates the supercoiling. Another one of our methods was to use a dCas9 clamp between the RFP and GFP to clamp down between the two genes and stop the supercoiling. In order to see whether or not the problem of supercoiling was reduced, we had to first construct the plasmid circuits used for our experiment by using Golden Gate Assembly. First, we designed primers to amplify our parts off current iGEM parts using the online DNA design editor, Benchling.For our experiment, we have used RFP and GFP and parts from the iGEM inventory: BBa_J04450 and BBa_I13522, respectively. These parts were amplified off and ligated together in varying orientations (see picture designs) with a spacer part between them (either 500bp spacer, 1000bp spacer, or a dcas9 clamp site spacer) and finally a vector, or backbone piece, with Chloramphenicol or Kanamycin resistance.
The RFP gene and the GFP gene with transcription occurring in opposite directions away from the dCas 9 binding sites, which separate the two genes.
The GFP gene being transcribed towards the dCas9 binding sites and the RFP gene, while the RFP gene is being transcribed away from the GFP gene.
The RFP gene and GFP gene while transcription is occurring towards each other, as well as in the direction of the dCas 9 binding sites.
The RFP gene being transcribed towards the dCas 9 binding sites and the GFP gene, while the GFP gene is being transcribed away from RFP gene.

The RFP gene and GFP gene are switched. The RFP and the GFP with transcription occurring in opposite directions away from the dCas 9 binding sites, which separate the two genes.
RFP and GFP are switched. The RFP gene being transcribed towards the dCas9 binding sites and the GFP gene while the GFP gene being transcribed away from the RFP gene.
RFP and GFP are switched. The RFP gene and GFP gene while transcription is occurring towards each other, as well as in the direction of the dCas 9 binding sites.
RFP and GFP are switched. The GFP gene being transcribed towards the dCas 9 binding sites and the RFP gene, while the RFP gene is being transcribed away from the GFP gene.