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| <div class="project-content applications"> | | <div class="project-content applications"> |
− | <h2><a name="apply">Applications</a></h2> | + | <h2><a name="apply">Part Improvement</a></h2> |
− | <p>In our project, for the measurement of our quorum sensing response, we used a system based on a reporter fluorescent protein (EYFP) and a control one (ECFP), constitutively expressed, that would be subject to the same extrinsic variation as the reporter gene, but would not respond to our tested stimuli, allowing us to use this CFP measurement to normalize YFP. This gives us the possibility of a much more reproducible result, less dependant on growth media and cell density (a rather unreliable parameter to measure) at the time of each measurement. | + | <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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− | Something else we did to raise the precision of our measurements was not using LB for measuring in the plate reader, as it had high autofluorescence and commonly exibited fluorescence higher than our tests at low CFP values. Also, LB may change its color depending on how old it is and other environmental interactions. By centrifuging the cells for each sample and resuspending them in molecular grade H2O, we had a blank with much lesser autofluorescence and with less variance between measurements.
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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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| + | <center><img src="https://static.igem.org/mediawiki/2018/3/38/T--USP-Brazil--Dual_Fluo_Spectrum.png" style="width:95%"></center> |
| + | </div> |
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| + | <center><img src="https://static.igem.org/mediawiki/2018/0/06/T--USP-Brazil--cfp.png.png"></center> |
| + | <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. |
| + | </p> |
| + | <center><img src="https://static.igem.org/mediawiki/2018/b/be/T--USP-Brazil--measurement_abstract.png" style="width:80%"></center> |
| + | <h3>References</h3> |
| + | <ul> |
| + | <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> |
| + | </ul> |
| + | </div> |
| </div> | | </div> |
| </article> | | </article> |