Team:UIUC-Illinois/Project/Design

From 2012.igem.org

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<center><img src="https://static.igem.org/mediawiki/2012/4/44/ControlPUF.png"></center>
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<p>In order to test whether the endonuclease activity is specific to the PUF binding site, we propose to "mismatch" the PUF-PIN protein with the *PUF-PIN binding site with a YFP reporter. This experiment will be compared to the fluorescence of a control 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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<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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<center><h2>Theoretical Results</h2></center><br/>
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<center><img src="https://static.igem.org/mediawiki/2012/7/75/TheoryResults.png"></center><br/>
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<p>The above chart shows our predicted results. The control binding sites are predicted to bind both wild and mutant PUF-PIN fusion proteins. The only non-expression combinations are when we match a PUF-PIN or *PUF-PIN to it's respective binding site which should result in endonuclease activity and subsequent silencing of the YFP reporter gene.</p><br/>
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Revision as of 04:44, 29 September 2012

Header

Project Design

Project Design

  • Overview
  • PUF+PIN Fusion
  • YFP Reporter
  • Non-Specific Control
  • Theoretical Results
  • 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.

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