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>
  
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<img border="0" class='img-responsive' width="500" src="https://static.igem.org/mediawiki/2015/7/7e/Manquante2.png" alt="" />
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<p class="text-justify"><strong> Figure 1 : Hypoxia inducible promoter</strong></p>
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<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>
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<img border="0" class='img-responsive' width="500" src="https://static.igem.org/mediawiki/2015/7/7e/Manquante2.png" alt="" />
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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>
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<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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<h2>Inducing apoptosis in hypoxic environment</h2>
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<p class="text-justify">We have a way to detect hypoxia area to locate tumor cells, now it’s time to destroy them.
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In order to kill hypoxic tumor cells, we created a yeast secretion system for perforin and granzyme B under control of our hypoxia bio-sensor. Perforin is a pore-forming enzyme, allowing granzyme B to enter the cell. Granzyme B is a serine protease capable of inducing cell apoptosis by caspase activation. Co-expressed, these enzymes have proven their potential to induce cancer cell death :</p>
  
  

Revision as of 23:57, 18 September 2015

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