Difference between revisions of "Team:EPF Lausanne/Notebook/Yeast"

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     <!--Integrate pDCAS9-->
 
     <!--Integrate pDCAS9-->
 
     <div id="integrate_pTPGI_dCas9_VP64" class="panel">
 
     <div id="integrate_pTPGI_dCas9_VP64" class="panel">
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     <h1><small>Integrate pTPGI_dCas9_VP64</small></br>Integrate pTPGI_dCas9_VP64</h1>
+
     <h1><small>Express dCas9-VP64</small></br>Integrate pTPGI_dCas9_VP64</h1>
 
         <p><small>We received plasmid pTPGI_dCas9_VP64 from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and miniprep, we performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid, in order to integrate it into yeast genome. For more details about our experiments, see <a target="_blank" href="https://static.igem.org/mediawiki/2015/3/30/EPF_Lausanne_Journal_integrate_dCas9-VP64.pdf">here</a>. Only our fourth trial to integrate the plasmid was successful.
 
         <p><small>We received plasmid pTPGI_dCas9_VP64 from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and miniprep, we performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid, in order to integrate it into yeast genome. For more details about our experiments, see <a target="_blank" href="https://static.igem.org/mediawiki/2015/3/30/EPF_Lausanne_Journal_integrate_dCas9-VP64.pdf">here</a>. Only our fourth trial to integrate the plasmid was successful.
 
         </small></p>
 
         </small></p>
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     <!--Integrate pDCAS9-->
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     <!--Western Blot pDCAS9-->
 
     <div id="wb_dCas9-VP64" class="panel">
 
     <div id="wb_dCas9-VP64" class="panel">
−
     <h1><small>Integrate pTPGI_dCas9_VP64</small></br>Integrate pTPGI_dCas9_VP64</h1>
+
     <h1><small>Express dCas9-VP64</small></br>Western Blot of dCas9-VP64</h1>
 
         <p><small>The Western Blot allows to check the expression of dCas9.
 
         <p><small>The Western Blot allows to check the expression of dCas9.
 
         </small></p>
 
         </small></p>

Revision as of 12:18, 19 August 2015

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

saccharomyces cerevisiae

Express dCas9-VP64
Integrate pTPGI_dCas9_VP64

We received plasmid pTPGI_dCas9_VP64 from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and miniprep, we performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid, in order to integrate it into yeast genome. For more details about our experiments, see here. Only our fourth trial to integrate the plasmid was successful.

Materials and method

  • Glycerol stocks
  • Miniprep
  • Restriction analysis
  • Polymerase Chain Reaction
  • Yeast integration A AJOUTER SUR PROTOCOLS

Results

We used four different sets of enzymes for the restriction analysis. Linearized pTPGI_dCas9_VP64 is expected to be 10'987 bp. We observe that the gel (fig.1) corresponds to the expected one.
The plasmid was linearised both with EagI HF and NotI HF prior to integration. We integrated each linearised plasmid to obtain two different yeast strains.

Express dCas9-VP64
Western Blot of dCas9-VP64

The Western Blot allows to check the expression of dCas9.

Materials and method

  • Western Blot A AJOUTER SUR PROTOCOLS

Results

Results of Western Blot.

Integrate reporter plasmid
Linearise reporter plasmid

We received plasmid pCYC_yeGFP in bacteria from Addgene. The plasmid was found from ... We inoculated single colonies of bacteria in order to prepare glycerol stocks and minipreps. We performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid by PCR, in order to integrate it into yeast genome.

Materials and method

  • Glycerol stocks
  • Miniprep
  • Restriction analysis
  • Polymerase Chain Reaction

Results

We used four different sets of enzymes for the restriction analysis. Linearized pCYC_yeGFP is expected to be 10'987 bp. We observe that the gel (fig.1) corresponds to the expected one (fig. 2).
The plasmid was linearised both with ... and ... prior to integration. We integrated each linearised plasmid to obtain two different yeast strains.

Integrate reporter plasmid
Synthesize promoters

The promoter CYC#0 was already present in plasmid pCYC_yeGFP from Addgene. We synthesized three other promoters CYC#1, CYC#2 and CYC#3. The only differences between one another were the three gRNA SDSs c3, c6 and c7.

Materials and method

  • Synthesis ??

Results

We obtain four different promoters CYC#0, CYC#1, CYC#2 and CYC#3 according to fig. ...

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits