Team:UIUC-Illinois/Results

From 2012.igem.org

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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.
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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.
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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/>
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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.
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As of now, we are still running more mcherry experiments. The data showed some preliminary results.  
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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.  
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Revision as of 05:44, 26 October 2012

Header

Data

PUF Experimental Results

  • Overview
  • Mutant PUF Expression Gel
  • YFP Fluorescence Results
  • mCherry Fluorescence Results
  • Conclusion
  • PUF 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.

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