Difference between revisions of "Team:Evry/Project/Biosensor"

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<p class="text-justify">HIF transcription factors are composed of two sub-units. The α-subunits of the HIF transcription factors are degraded by proteasomal pathways during normoxia but are stabilized under hypoxic conditions (6). On the contrary, the beta-subunit of HIF is always expressed and maintained stable in the cytosol. We codon optimized the human HIF-alpha and HIF-beta for yeast and cloned these proteins in yeast S. cerevisiae under control of GAL1, a galactose inducible promoter.</p>
 
<p class="text-justify">HIF transcription factors are composed of two sub-units. The α-subunits of the HIF transcription factors are degraded by proteasomal pathways during normoxia but are stabilized under hypoxic conditions (6). On the contrary, the beta-subunit of HIF is always expressed and maintained stable in the cytosol. We codon optimized the human HIF-alpha and HIF-beta for yeast and cloned these proteins in yeast S. cerevisiae under control of GAL1, a galactose inducible promoter.</p>
  
<img border="0" class='img-responsive' width="500" src="https://static.igem.org/mediawiki/2015/4/47/Biosensor_schema.png" alt="" />
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<center><img border="0" class='img-responsive' width="500" src="https://static.igem.org/mediawiki/2015/4/47/Biosensor_schema.png" alt="" />
<p class="text-justify"><strong> Figure 1 : Hypoxia inducible promoter</strong></p>
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<p class="text-justify"><strong> Figure 1 : Hypoxia inducible promoter</strong></p></center>
  
 
<p class="text-justify">To measure the activity of the bio-sensor, the gene reporter RFP was expressed under control of this HRE-CMV promoter. We used cobalt, a transition metal, to induce hypoxia. Cobalt mimics hypoxia by causing the stabilization of HIF-α (7).</p>
 
<p class="text-justify">To measure the activity of the bio-sensor, the gene reporter RFP was expressed under control of this HRE-CMV promoter. We used cobalt, a transition metal, to induce hypoxia. Cobalt mimics hypoxia by causing the stabilization of HIF-α (7).</p>
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<p class="text-justify">Cloning steps were successful as showed sequencing data and colony PCR (figure 2). To remove the promoter GAL1 from our plasmid pYGG1 and replace it with the HRE/CMV promoter, we performed site-directed mutagenesis. First, we amplified the plasmid around GAL1 to remove it. Then, we phospholyrated with a kinase the blunt ends, we added DpnI to remove the template DNA and we ligated with T4 kinase. Colony PCR followed by digestion confirmed the successful assembly by golden gate:</p>
 
<p class="text-justify">Cloning steps were successful as showed sequencing data and colony PCR (figure 2). To remove the promoter GAL1 from our plasmid pYGG1 and replace it with the HRE/CMV promoter, we performed site-directed mutagenesis. First, we amplified the plasmid around GAL1 to remove it. Then, we phospholyrated with a kinase the blunt ends, we added DpnI to remove the template DNA and we ligated with T4 kinase. Colony PCR followed by digestion confirmed the successful assembly by golden gate:</p>
  
<img border="0" class='img-responsive' width="500" src="https://static.igem.org/mediawiki/2015/a/a2/Gel_jpg.jpg " alt="" />
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<center><img border="0" class='img-responsive' width="500" src="https://static.igem.org/mediawiki/2015/a/a2/Gel_jpg.jpg " alt="" />
<p class="text-justify"><strong> Figure 2: Colony PCR for biosensor 2 (HIF alpha and beta) and for biosensor 3 (HRE-CMV-RFP)</strong></p>
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<p class="text-justify"><strong> Figure 2: Colony PCR for biosensor 2 (HIF alpha and beta) and for biosensor 3 (HRE-CMV-RFP)</strong></p></center>
  
 
<p class="text-justify">Due to yeast autofluorescence in the red channel, we were not able to perform a fluorescence measurement with the Red Fluorescent Protein We will further characterize our bio-sensor with a LacZ assay instead of RFP. </p>
 
<p class="text-justify">Due to yeast autofluorescence in the red channel, we were not able to perform a fluorescence measurement with the Red Fluorescent Protein We will further characterize our bio-sensor with a LacZ assay instead of RFP. </p>
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</ul>
 
</ul>
  
<img border="0" class='img-responsive' width="500" src="https://static.igem.org/mediawiki/2015/e/e0/Biosensor2.jpg " alt="" />
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<center><img border="0" class='img-responsive' width="500" src="https://static.igem.org/mediawiki/2015/e/e0/Biosensor2.jpg " alt="" />
<p class="text-justify"><strong>Figure 3 : Yeast encapsulated secreting perforine and granzyme B upon hypoxia detection</strong></p>
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<p class="text-justify"><strong>Figure 3 : Yeast encapsulated secreting perforine and granzyme B upon hypoxia detection</strong></p></center>
  
 
<p class="text-justify">For optimal conditions of translation, we placed both proteins on the same mRNA under control of the CMV minimal promoter. The Internal Ribosome Entry Site IRES URE2, effective in yeast, was placed ahead of the second protein to ensure two translations on the same mRNA.</p>
 
<p class="text-justify">For optimal conditions of translation, we placed both proteins on the same mRNA under control of the CMV minimal promoter. The Internal Ribosome Entry Site IRES URE2, effective in yeast, was placed ahead of the second protein to ensure two translations on the same mRNA.</p>

Revision as of 01:45, 19 September 2015

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