Difference between revisions of "Team:Cologne-Duesseldorf/Improve"

 
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<h1>Improve</h1>
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      <h3>★  ALERT! </h3>
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      <p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
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      <p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2017.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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We are proud to improve roGFP(BBa_K1093013) of Team iGEM13_Warsaw with our part (BBa_K2271024).
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In order to use the redox sensing <i>in vivo</i> sensor we optimized it for our application in yeast. Carboxyl terminal fusion of the peroxisomal targeting signal 1 (PTS1) resulted in small areas with high fluorescence intensity inside the cell - proven to be <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Results">peroxisomes</a>.
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In contrast, cytosolic fluorescence was nearly silenced. The Pex5 receptor recognizes the signaling tag PTS1 and ensures the import into the peroxisome, which is shown in the following figure.
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<font size="3"> Right: localisation of roGFP2 into the peroxisomes, Left: wild type control </font>
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<p>Expression of our level2 plasmids containing both, a sensor and peroxin13 mRuby, a peroxisomal marker, showed co-localization which leaded to yellow spots in merged channels
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<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655559/"> <abbr title="2009, Robert Yung-Liang Wang et al.- A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes">
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<font size ="3"<Robert Yung-Liang Wang <i>et al.</i>, (2009)</font></abbr> </a>.</p>
  
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<figcaption><font size="3"> Right: peroxin13-mruby and cytosolic expressed roGFP2, Left: PTS1 tagged roGFP2 co-localized with peroxin13-mruby</font> </figcaption>
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We successfully validated roGFP2 and conducted calibration and <i>in vivo</i> measurements.
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Latest revision as of 22:25, 1 November 2017

Improve

We are proud to improve roGFP(BBa_K1093013) of Team iGEM13_Warsaw with our part (BBa_K2271024). In order to use the redox sensing in vivo sensor we optimized it for our application in yeast. Carboxyl terminal fusion of the peroxisomal targeting signal 1 (PTS1) resulted in small areas with high fluorescence intensity inside the cell - proven to be peroxisomes. In contrast, cytosolic fluorescence was nearly silenced. The Pex5 receptor recognizes the signaling tag PTS1 and ensures the import into the peroxisome, which is shown in the following figure.

Right: localisation of roGFP2 into the peroxisomes, Left: wild type control

Expression of our level2 plasmids containing both, a sensor and peroxin13 mRuby, a peroxisomal marker, showed co-localization which leaded to yellow spots in merged channels et al., (2009) .

Right: peroxin13-mruby and cytosolic expressed roGFP2, Left: PTS1 tagged roGFP2 co-localized with peroxin13-mruby

We successfully validated roGFP2 and conducted calibration and in vivo measurements.