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From 2012.igem.org

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-	<center><h2>Experimental Results</h2></center>	+	<center><h2>PUF Experimental Results</h2></center>
	<li><a name="data0" >Overview</a></li>		<li><a name="data0" >Overview</a></li>
-	<li><a name="data1" >Mutant PUF Expression Gel</a></li>	+
	<li><a name="data2" >YFP Fluorescence Results</a></li>		<li><a name="data2" >YFP Fluorescence Results</a></li>
	<li><a name="data3" >mCherry Fluorescence Results</a></li>		<li><a name="data3" >mCherry Fluorescence Results</a></li>
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	<div id="dataOverview">		<div id="dataOverview">
-	<center><h2>Results Overview</h2></center>	+	<center><h2>PUF Results Overview</h2></center>
	<br/>		<br/>
-	<p>The results covered in this section are of the experiments overviewed in the <a href="https://2012.igem.org/Team:UIUC-Illinois/Project/Design">Design section</a>. Please note that not all the experiments have been completed or have data ready for disclosure. The data that is complete, that has been collected and compiled, is listed to the left for their respective experiments.</p>	+	<p>The results covered in this section are of the experiments overviewed in the <a href="https://2012.igem.org/Team:UIUC-Illinois/Project/Design">Design section</a>. Please note that not all planned experiments have been completed or have data ready for disclosure. The data that is complete, that which has been collected and compiled, is listed to the left for their respective experiments.</p>
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	<center><h2>Results Overview</h2></center>		<center><h2>Results Overview</h2></center>
	<br/>		<br/>
-	<p>The results covered in this section are of the experiments overviewed in the <a href="https://2012.igem.org/Team:UIUC-Illinois/Project/Design">Design section</a>. Please note that not all the experiments have been completed or have data ready for disclosure. The data that is complete, that has been collected and compiled, is listed to the left for their respective experiments.</p>	+	<p>The results covered in this section are of the experiments overviewed in the <a href="https://2012.igem.org/Team:UIUC-Illinois/Project/Design">Design section</a>. Please note that not all planned experiments have been completed or have data ready for disclosure. The data that is complete, that which has been collected and compiled, is listed to the left for their respective experiments.</p>
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	<center><h2>YFP Fluorescence Data</h2></center><br/>		<center><h2>YFP Fluorescence Data</h2></center><br/>
-	<img src="https://static.igem.org/mediawiki/2012/5/50/YFPFluorescence.png" height=400 width=715>	+	<img src="https://static.igem.org/mediawiki/2012/d/d2/FixedYFPfltest.png" height=400 width=715>
	<br/><br/>		<br/><br/>
-	With regards to our hypothetical results, our experimental fluorescence measurements were substantially faithful to our predictions. Looking at columns 5, 6, 7, and 8, our observations appear to be in line with what we have predicted in introducing the wild type PUF-PIN to constructs containing a control recognition site and a specific recognition site. Columns 1 and 2 are negative fluorescence controls while Columns 3 and 4 are positive fluorescence controls measured without the influence of any sort of binding site.	+	With regards to our hypothetical results, our experimental fluorescence measurements were substantially faithful to our predictions. Looking at columns 5, 6, 7, and 8, our observations appear to be in line with what we have predicted in introducing the wild type PUF-PIN to constructs containing specific recognition site. However, YFP expression was silenced regardless of the binding site type present in the YFP expression constructs. With many control experiments and repeated tests, we thought the most possible reason could be that PUF recognizes sequences in the YFP gene (although the YFP sequences provided from the registry showed no PUF binding sites <a href="http://partsregistry.org/cgi/partsdb/puttext.cgi">in silico</a>). Columns 1 and 2 are negative fluorescence controls while Columns 3, 4, 5, and 6 are positive fluorescence controls.
	<br/><br/>		<br/><br/>
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	<center><h2>mCherry Fluorescence Data</h2></center><br/>		<center><h2>mCherry Fluorescence Data</h2></center><br/>
-	<img src="https://static.igem.org/mediawiki/2012/7/75/RFPFluorescence.png" height=450 width=715>	+	<img src="https://static.igem.org/mediawiki/2012/f/f3/NEWmcherry.png" height=100% width=100%>
	<br/><br/>		<br/><br/>
	In order to facilitate more reliable results, we also tested our constructs with the expression of an RFP, mCherry. If in the case that confounding factors influenced our results in our experiments using YFP, we could minimize inaccurate influences on our data by using a different reporter. <br/><br/>		In order to facilitate more reliable results, we also tested our constructs with the expression of an RFP, mCherry. If in the case that confounding factors influenced our results in our experiments using YFP, we could minimize inaccurate influences on our data by using a different reporter. <br/><br/>
-	However, this experiment (though not as intensively compared with numerous controls as our YFP tests) has yielded unexpected results. Suggesting key differences in the gene silencing ability of PUF when used with mCherry on a Protet plasmid. Though this is not what we expected, these results are still somewhat inconclusive since we did not test the same construct on a pBAD30 plasmid instead of Protet. pBAD30 is what we used for our YFP + binding site + wild type PUF-PIN fluorescence experiments. Further experimentation is underway.	+	As of now, we are still running more mcherry experiments. The data showed some preliminary results.
		+	The results confirmed our hypothesis that PUF-PIN don’t silence the gene from the control construct. This confirms our hypothesis that PUF-PIN doesn't silence the gene from the control construct.
	</div>		</div>
	<div id="data4" style="display:none">		<div id="data4" style="display:none">
-	<center><h2>Conclusion</h2></center><br/><br/>	+	<center><h2>Conclusion</h2></center><br/>
		+	<p>The YFP fluorescence data has shown that the PUF-PIN domain has the ability to silence YFP expression. However, YFP expression was silenced regardless of the binding site type present in the YFP expression constructs. Assessing the specificity of PUF requires further experimental research. The mCherry data suggests that it is likely due to a problem with the YFP reporter, but due to the fact that there was no control binding site cloned into the mCherry expression construct, more experiments will be required for conclusive evidence. The specificity and customizability of our gene silencing through RNA scission both have extensive ongoing experiments.</p>
	</div>		</div>
	</div>		</div>
	</div>		</div>

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Latest revision as of 21:10, 26 October 2012

Results Overview

The results covered in this section are of the experiments overviewed in the Design section. Please note that not all planned experiments have been completed or have data ready for disclosure. The data that is complete, that which has been collected and compiled, is listed to the left for their respective experiments.

Mutant PUF Expression Gel

This is our PUF protein expression gel. We decided to test the PUF protein expression under different conditions to optimize it our further experiments. Lanes labeled 2 through 7 were expressed in a higher, E. Coli standard temperature of 37 degrees C. Lanes 8 through 13 were conducted in a lower temperature of 24 degrees C. The lower temperature, as suggested by the Zefang Wang Lab at the University of North Carolina, has been confirmed to result in more specific and definite PUF protein expression in DH5a.

The parts highlighted in red are significant expressions of the wild type PUF protein.

YFP Fluorescence Data

With regards to our hypothetical results, our experimental fluorescence measurements were substantially faithful to our predictions. Looking at columns 5, 6, 7, and 8, our observations appear to be in line with what we have predicted in introducing the wild type PUF-PIN to constructs containing specific recognition site. However, YFP expression was silenced regardless of the binding site type present in the YFP expression constructs. With many control experiments and repeated tests, we thought the most possible reason could be that PUF recognizes sequences in the YFP gene (although the YFP sequences provided from the registry showed no PUF binding sites in silico). Columns 1 and 2 are negative fluorescence controls while Columns 3, 4, 5, and 6 are positive fluorescence controls.

As a reference to our aformentioned experimental design predictions. The theoretical results chart is shown here again.

mCherry Fluorescence Data

In order to facilitate more reliable results, we also tested our constructs with the expression of an RFP, mCherry. If in the case that confounding factors influenced our results in our experiments using YFP, we could minimize inaccurate influences on our data by using a different reporter.

As of now, we are still running more mcherry experiments. The data showed some preliminary results. The results confirmed our hypothesis that PUF-PIN don’t silence the gene from the control construct. This confirms our hypothesis that PUF-PIN doesn't silence the gene from the control construct.

Conclusion

The YFP fluorescence data has shown that the PUF-PIN domain has the ability to silence YFP expression. However, YFP expression was silenced regardless of the binding site type present in the YFP expression constructs. Assessing the specificity of PUF requires further experimental research. The mCherry data suggests that it is likely due to a problem with the YFP reporter, but due to the fact that there was no control binding site cloned into the mCherry expression construct, more experiments will be required for conclusive evidence. The specificity and customizability of our gene silencing through RNA scission both have extensive ongoing experiments.