Team:UIUC-Illinois/Project/Design

From 2012.igem.org

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<p>In designing our project we based our quantitative tests on fluorescence measured by a plate fluorescence reader. Our constructs were created in ways that best suit providing evidence for our hypothesis of the PUF-PIN fusion protein showing endonuclease activity. Generally, our results were collected from quantifying two main PUF-PIN fusion protein types, a wild type and a mutant type, in different conditions. These two fusion proteins had recognition sites that differed by one base pair. <br/><br/>Click on the list to the left to read about each of our constructs and why we decided to do them.</p>
<p>In designing our project we based our quantitative tests on fluorescence measured by a plate fluorescence reader. Our constructs were created in ways that best suit providing evidence for our hypothesis of the PUF-PIN fusion protein showing endonuclease activity. Generally, our results were collected from quantifying two main PUF-PIN fusion protein types, a wild type and a mutant type, in different conditions. These two fusion proteins had recognition sites that differed by one base pair. <br/><br/>Click on the list to the left to read about each of our constructs and why we decided to do them.</p>
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<p>The light blue symbol labeled *PUF-PIN represents the gene that is expressed to produce a mutant-type PUF fused to a PIN endonuclease. The variation between our two constructs is only a single base pair difference between the 8-base pair PUF-PIN and *PUF-PIN RNA recognition sites. Otherwise, their endonuclease function is theoretically unaffected by the different recognition subunits. </p><br/>
<p>The light blue symbol labeled *PUF-PIN represents the gene that is expressed to produce a mutant-type PUF fused to a PIN endonuclease. The variation between our two constructs is only a single base pair difference between the 8-base pair PUF-PIN and *PUF-PIN RNA recognition sites. Otherwise, their endonuclease function is theoretically unaffected by the different recognition subunits. </p><br/>
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Since our YFP reporter gene is located downstream of the recognized PUF-PIN cut site, we would be able to quantify changes in expression and fluorescence with and without the introduction of our specific RNA binding endonuclease activity. We hypothesized that in measuring the fluorescence levels, we would have evidence supporting PUF-PIN and *PUF-PIN as RNA scissors with the ability to silence genes.  
Since our YFP reporter gene is located downstream of the recognized PUF-PIN cut site, we would be able to quantify changes in expression and fluorescence with and without the introduction of our specific RNA binding endonuclease activity. We hypothesized that in measuring the fluorescence levels, we would have evidence supporting PUF-PIN and *PUF-PIN as RNA scissors with the ability to silence genes.  
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<p>In order to test whether the endonuclease activity is specific to the PUF binding site, we propose to match both the PUF-PIN and *PUF-PIN proteins with a controlled wild/mutant binding site with a YFP reporter. This experiment will be compared to the fluorescence of a PUF-PIN protein matched with it's PUF-PIN binding site with YFP reporter, YFP reporter constructs alone, and other such comparative constructs under different conditions like temperature to determine optimal experimental conditions. </p>
<p>In order to test whether the endonuclease activity is specific to the PUF binding site, we propose to match both the PUF-PIN and *PUF-PIN proteins with a controlled wild/mutant binding site with a YFP reporter. This experiment will be compared to the fluorescence of a PUF-PIN protein matched with it's PUF-PIN binding site with YFP reporter, YFP reporter constructs alone, and other such comparative constructs under different conditions like temperature to determine optimal experimental conditions. </p>
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Revision as of 08:37, 3 October 2012

Header

Project Design

PUF Project Design

  • Overview
  • PUF+PIN Fusion
  • YFP Reporter
  • Non-Specific Control
  • Reliability experiments
  • Theoretical Results
  • PUF Experimental Design


    In designing our project we based our quantitative tests on fluorescence measured by a plate fluorescence reader. Our constructs were created in ways that best suit providing evidence for our hypothesis of the PUF-PIN fusion protein showing endonuclease activity. Generally, our results were collected from quantifying two main PUF-PIN fusion protein types, a wild type and a mutant type, in different conditions. These two fusion proteins had recognition sites that differed by one base pair.

    Click on the list to the left to read about each of our constructs and why we decided to do them. All expressions were done in vivo with the DH5a strain of E.Coli on pBAD30 or Protet plasmids.

    Retrieved from "http://2012.igem.org/Team:UIUC-Illinois/Project/Design"