Difference between revisions of "Team:USP-Brazil/Improve"

 
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{{USP-Brazil/PROJECT}}
 
{{USP-Brazil/PROJECT}}
 
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                      <a href="https://2018.igem.org/Team:USP-Brazil/Overview">Overview</a>
 
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                      <a href="https://2018.igem.org/Team:USP-Brazil/Results">Results</a>
 
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                      <a href="https://2018.igem.org/Team:USP-Brazil/Part_Collection">Collection of Parts</a>
 
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           <article>
 
           <article>
 
           <div class="project-content applications">
 
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             <h2><a name="apply">Applications</a></h2>
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             <h2><a name="apply">Part Improvement</a></h2>
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             <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec interdum lacinia posuere. Sed nunc felis, consequat a tincidunt eu, lacinia id nisi. Praesent eget erat consequat, ultrices justo nec, eleifend ex. Donec iaculis turpis id augue molestie, in venenatis enim pellentesque. Etiam quis semper diam, sit amet rhoncus libero. Vivamus pulvinar bibendum lacus, at iaculis enim tempor semper. In ac enim libero. Mauris at porta mi, in euismod arcu.
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             <p>We improved the part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_E0022">BBa_E0430</a>, an EYFP output device, by joining it with a constitutively expressed (by ptet, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_R0040">BBa_R0040</a>) ECFP gene, to be used as a control for normalization of the output measurement, as this control gene would be subject to the same influences as the reporter gene (eg. cell density, plasmid copy number, metabolic load, environment properties). Instead of dividing the YFP measurement by the OD, as usual, dividing the construct's YFP output by the CFP output gives us better precision and an adimensional value for promoter strength. This method was used following the efforts of Rudge et al. on characterizing intrinsic promoter properties.
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             </p>
 
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                <p>This fluorescent protein pair is good for this type of measurement due to their spectral separation. In our experiments, we used excitation/emission values of 430/480 for CFP and 500/530 for YFP. The CFP protein has an LVA degradation tag, which should make the CFP measure more accurate as a representation of the immediate state of the gene's activity, independing of non-degraded, previous values.
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                </p>
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                <center><img src="https://static.igem.org/mediawiki/2018/3/38/T--USP-Brazil--Dual_Fluo_Spectrum.png" style="width:95%"></center>
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              <center><img src="https://static.igem.org/mediawiki/2018/0/06/T--USP-Brazil--cfp.png.png"></center>
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              <p>Unfortunately, this makes the CFP overall fluorescence lower and creates the need for a more precise measurement, rather than just measuring from LB. We have obtained good results while using LB and centrifuging the samples then ressuspending them in H2O, or while growing the cells in M9 medium.
 +
              </p>
 +
              <p>Our new part is <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2771020">BBa_K2771020</a>, which still preserves de modularity property of the original part, as other teams can insert promoter parts upstream of our part and readily have a good reporter gene for the activity of one promoter. As we showed in our <a href="https://2018.igem.org/Team:USP-Brazil/Measurement">Measurement</a> page, this method also allows for a more constant value for controls and constitutive expression, having a smaller standard deviation relative to the mean, when compared to the same data when using OD as a normalization factor. We also built parts using quorum sensing promoters and characterized their activity using this improvement.
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              </p>
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              <center><img src="https://static.igem.org/mediawiki/2018/b/be/T--USP-Brazil--measurement_abstract.png" style="width:80%"></center>
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              <h3>References</h3>
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              <ul>
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                <li>T.J.Rudge, J.R.Brown, F.Federici, N.Dalchau, A.Phillips, J.W.Ajioka, J.Haseloff. “Characterization of Intrinsic Properties of Promoters” ACS Synthetic Biology 5 (1), 89-98, (2016) doi:10.1021/acssynbio.5b00116  </li>
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              </ul>
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{{USP-Brazil/FOOTER}}

Latest revision as of 01:54, 18 October 2018

Wiki - iGEM Brazil

Part Improvement

We improved the part BBa_E0430, an EYFP output device, by joining it with a constitutively expressed (by ptet, BBa_R0040) ECFP gene, to be used as a control for normalization of the output measurement, as this control gene would be subject to the same influences as the reporter gene (eg. cell density, plasmid copy number, metabolic load, environment properties). Instead of dividing the YFP measurement by the OD, as usual, dividing the construct's YFP output by the CFP output gives us better precision and an adimensional value for promoter strength. This method was used following the efforts of Rudge et al. on characterizing intrinsic promoter properties.

This fluorescent protein pair is good for this type of measurement due to their spectral separation. In our experiments, we used excitation/emission values of 430/480 for CFP and 500/530 for YFP. The CFP protein has an LVA degradation tag, which should make the CFP measure more accurate as a representation of the immediate state of the gene's activity, independing of non-degraded, previous values.

Unfortunately, this makes the CFP overall fluorescence lower and creates the need for a more precise measurement, rather than just measuring from LB. We have obtained good results while using LB and centrifuging the samples then ressuspending them in H2O, or while growing the cells in M9 medium.

Our new part is BBa_K2771020, which still preserves de modularity property of the original part, as other teams can insert promoter parts upstream of our part and readily have a good reporter gene for the activity of one promoter. As we showed in our Measurement page, this method also allows for a more constant value for controls and constitutive expression, having a smaller standard deviation relative to the mean, when compared to the same data when using OD as a normalization factor. We also built parts using quorum sensing promoters and characterized their activity using this improvement.

References

  • T.J.Rudge, J.R.Brown, F.Federici, N.Dalchau, A.Phillips, J.W.Ajioka, J.Haseloff. “Characterization of Intrinsic Properties of Promoters” ACS Synthetic Biology 5 (1), 89-98, (2016) doi:10.1021/acssynbio.5b00116