Team:UIUC-Illinois/Project/Design

From 2012.igem.org

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                     <li><a name="des2" >YFP Reporter</a></li>
                     <li><a name="des2" >YFP Reporter</a></li>
                     <li><a name="des3" >Non-Specific Control</a></li>
                     <li><a name="des3" >Non-Specific Control</a></li>
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                     <li><a name="des4" >Reliability experiments</a></li>
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                     <li><a name="des4" >Experiments</a></li>
                     <li><a name="des5" >Theoretical Results</a></li>
                     <li><a name="des5" >Theoretical Results</a></li>
<center><img src="http://2012.igem.org/wiki/images/4/47/Legend2.png" width=100%></center>
<center><img src="http://2012.igem.org/wiki/images/4/47/Legend2.png" width=100%></center>
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<center><img src="http://2012.igem.org/wiki/images/e/e1/PUFPINwild.png" alt="PUFPINwild" border=1></center><br/>
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<center><img src="http://2012.igem.org/wiki/images/4/4f/PUFPINwild2.png" alt="PUFPINwild" border=1></center><br/>
<p>The blue symbol labeled PUF-PIN represents the gene that is expressed to produce a wild-type PUF fused to a PIN endonuclease. The comparison of this construct's results to the mutant PUF-PIN (labeled mPUF-PIN) our main source of experimental data.</p><br/>
<p>The blue symbol labeled PUF-PIN represents the gene that is expressed to produce a wild-type PUF fused to a PIN endonuclease. The comparison of this construct's results to the mutant PUF-PIN (labeled mPUF-PIN) our main source of experimental data.</p><br/>
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<center><img src="http://2012.igem.org/wiki/images/a/a8/PUFPINmutant.png" alt="PUFPINmutant" border=1></center><br/>
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<center><img src="http://2012.igem.org/wiki/images/f/fc/PUFPINmutantNEW2.png" alt="PUFPINmutant" border=1></center><br/>
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<center><img src="http://2012.igem.org/wiki/images/b/bf/PUFPINreporter.png" border=1>
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<center><img src="http://2012.igem.org/wiki/images/6/61/Fixedwt.png" border=1>
<br/>Fig. 1</center><br/>
<br/>Fig. 1</center><br/>
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<center><img src="http://2012.igem.org/wiki/images/d/dc/PUFPINmutantreporter.png" border=1>
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<center><img src="http://2012.igem.org/wiki/images/1/10/Fixedmutant.png" border=1>
<br/>Fig. 2</center><br/>
<br/>Fig. 2</center><br/>
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<br/>Fig. 3</center><br/>
<br/>Fig. 3</center><br/>
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<center><img src="http://2012.igem.org/wiki/images/8/85/PUFPINmutantreporterCUT.png" border=1>
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<center><img src="http://2012.igem.org/wiki/images/8/85/PUFPINmutantreporterCUTNEW.png" border=1>
<br/>Fig. 4</center><br/>
<br/>Fig. 4</center><br/>
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<center><img src="http://2012.igem.org/wiki/images/4/44/ControlPUF.png" border=1></center>
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<center><img src="http://2012.igem.org/wiki/images/5/51/NewControlConstruct.png" border=1></center>
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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 mPUF-PIN proteins with a controlled wild/mutant binding site with a YFP reporter. The control YFP reporter construct encodes a binding site that doesnt recognize both wtPUF/mPUF protein.</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 mPUF-PIN proteins with a controlled wild/mutant binding site with a YFP reporter. The control YFP reporter construct encodes a binding site that doesnt recognize both wtPUF/mPUF protein.</p>
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<div id="des4" style="display:none">
<div id="des4" style="display:none">
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<center><h2>Reliability Experiments</h2></center><br/>
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<center><h2>Experiments</h2></center><br/>
<p>In order to eliminate the possibility of confounding factors affecting our apparent results, PUF-PIN experimental results were further reinforced with tests designed to consider other factors of our constructs possibly affecting our quantitative and qualitative measurements. Along with basic experiments testing our proposed constructs we also tested several controls relative to each of the different pieces of our constructs including the following:<br/><br/>
<p>In order to eliminate the possibility of confounding factors affecting our apparent results, PUF-PIN experimental results were further reinforced with tests designed to consider other factors of our constructs possibly affecting our quantitative and qualitative measurements. Along with basic experiments testing our proposed constructs we also tested several controls relative to each of the different pieces of our constructs including the following:<br/><br/>
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     <tr>
     <tr>
       <td>YFP + Control Binding Site in protet plasmid</td>
       <td>YFP + Control Binding Site in protet plasmid</td>
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       <td>This is a theoretically uninhibited YFP + binding site construct to determine the fluorescence of using YFP with a control binding site.</td>
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       <td>This is a positive control for the YFP experiments.</td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td>YFP + Specific Binding Site in protet plasmid</td>
       <td>YFP + Specific Binding Site in protet plasmid</td>
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       <td>Also a theoretically uninhibited YFP + binding site construct to determine the fluorescence of using YFP with a specific binding site</td>
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       <td>This is a positive control for the YFP experiments.</td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td>YFP + Control Binding Site in protet plasmid + pBAD30 Plasmid</td>
       <td>YFP + Control Binding Site in protet plasmid + pBAD30 Plasmid</td>
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       <td>This determines the effects of a pBAD30 Plasmid when used with a YFP + Control Binding Site.</td>
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       <td>This can help understand whether cotransformation of two plasmids in the cell will interfere with YFP expression.</td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td>YFP + Specific Binding Site in protet plasmid + pBAD30 Plasmid</td>
       <td>YFP + Specific Binding Site in protet plasmid + pBAD30 Plasmid</td>
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       <td>This determines the effects of a pBAD30 Plasmid when used with a YFP + Specific Binding Site.</td>
+
       <td>This can help understand whether cotransformation of two plasmids in the cell will interfere with YFP expression.</td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td>YFP + Specific Binding Site in protet plasmid + wild type PUF-PIN in pBAD30 plasmid</td>
       <td>YFP + Specific Binding Site in protet plasmid + wild type PUF-PIN in pBAD30 plasmid</td>
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       <td>This is a theoretically positive fluorescence control due to the endonuclease activity of a specifically bound PUF-PIN protein silencing the YFP gene.</td>
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       <td>This is a theoretically negative fluorescence control due to the endonuclease activity of a specifically bound PUF-PIN protein silencing the YFP gene.</td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td>YFP + Control Binding Site in protet plasmid + wild type PUF-PIN in pBAD30 plasmid</td>
       <td>YFP + Control Binding Site in protet plasmid + wild type PUF-PIN in pBAD30 plasmid</td>
-
       <td>This is a theoretically negative fluorescence control due to the endonuclease activity of a Control Binding Site bound PUF-PIN protein silencing the YFP gene.</td>
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       <td>This is a theoretically positive fluorescence control due to the endonuclease activity of a Control Binding Site bound PUF-PIN protein silencing the YFP gene.</td>
     </tr>
     </tr>
<tr><td>mCherry + Control Binding Site in protet plasmid </td>
<tr><td>mCherry + Control Binding Site in protet plasmid </td>
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<td>In order to minimize confounding factors coming from the YFP reporter itself, we tested our construct with a replaced RFP reporter as well</td></tr>
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<td>This is a positive control for the mCherry experiment</td></tr>
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<tr><td>mCherry + Control Binding Site in Protet plasmid + wild type PUF-PIN in pBAD30 plasmid</td>
+
 
-
<td>This test was to test the effects of the PUF-PIN endonuclease activity on a reporter other than YFP to pinpoint possible problems stemming from a YFP reporter</td>
+
-
</tr>
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     <tr>
     <tr>
       <td>mCherry + Wild type Binding Site in protet plasmid</td>
       <td>mCherry + Wild type Binding Site in protet plasmid</td>
-
       <td></td>
+
       <td>This is a positive control for the mCherry experiment.</td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td>mCherry + Mutant Binding Site in protet plasmid</td>
       <td>mCherry + Mutant Binding Site in protet plasmid</td>
-
       <td></td>
+
       <td>This is a positive control for the mCherry experiment.</td>
     </tr>
     </tr>
 +
 +
 +
<tr><td>mCherry + Control Binding Site in Protet plasmid + wild type PUF-PIN in pBAD30 plasmid</td>
 +
<td>This test was to test the effects of the PUF-PIN endonuclease activity on a reporter other than YFP to pinpoint possible problems stemming from a YFP reporter. Theoretically, we will see fluorescence.</td>
 +
</tr>
 +
 +
    <tr>
 +
      <td>mCherry + Control Binding Site in Protet plasmid+ mutant PUF-PIN in pBAD30 plasmid</td>
 +
      <td>This test is to show the specificity of mutant PUF-PIN. Theoretically, we will see fluorescence. </td>
 +
    </tr>
 +
     <tr>
     <tr>
       <td>mCherry + Specific Binding Site in Protet plasmid+ wild type PUF-PIN in pBAD30 plasmid</td>
       <td>mCherry + Specific Binding Site in Protet plasmid+ wild type PUF-PIN in pBAD30 plasmid</td>
-
       <td></td>
+
       <td>This test was to test the effects of the PUF-PIN endonuclease activity on a reporter other than YFP to pinpoint possible problems stemming from a YFP reporter</td>
     </tr>
     </tr>
 +
 +
     <tr>
     <tr>
       <td>mCherry + Specific Binding Site in Protet plasmid+ mutant PUF-PIN in pBAD30 plasmid</td>
       <td>mCherry + Specific Binding Site in Protet plasmid+ mutant PUF-PIN in pBAD30 plasmid</td>
-
       <td></td>
+
       <td>This test is to show mutant PUF-PIN's functionality. Theoretically, no fluorescence will be observed. </td>
-
    </tr>
+
-
    <tr>
+
-
      <td>mCherry + Control Binding Site in Protet plasmid+ mutant PUF-PIN in pBAD30 plasmid</td>
+
-
      <td></td>
+
     </tr>
     </tr>
 +
   </tbody>
   </tbody>
</table>
</table>

Latest revision as of 21:45, 26 October 2012

Header

Project Design

PUF Project Design

  • Overview
  • PUF+PIN Fusion
  • YFP Reporter
  • Non-Specific Control
  • 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 two base pairs.

    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 pPROTet.E plasmids.

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