Difference between revisions of "Part:BBa K1800001"
(→‎Team INSA UPS France 2017's usage with Pichia pastoris strain to secret Antimicrobial peptides)
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<p>The effect of signal factor was investigated by a toxicity assay with AMP.  We intend to see an inhibition effect of the yeast solution if the AMP are secreted i.e. if the alpha secretion factor works properly.  
 
<p>The effect of signal factor was investigated by a toxicity assay with AMP.  We intend to see an inhibition effect of the yeast solution if the AMP are secreted i.e. if the alpha secretion factor works properly.  
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Here is a summary of our result :   
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<p> Here is a summary of our result:   
  
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The engineered yeast were used in a halo assay against V. harveyi as the target of AMPs. A paper soacked with a yeast solution was placed on the plate and V. harveyi growth in the viscinity of the yeast patch was followed:
  
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figure coming soon
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/5/5c/Halos_pichia.png" width = "500"/><figcaption> Figure <b>AMP halo assay</b> Positive control is performed with chloramphenicol (25 g/L), the negative control is performed with empty plasmid integrated in P. pastoris, the assay is performed with plasmid containing BBa_K2278021 integrated in P. pastoris/ The yeast solution was concentrated 10 times</figcaption></figure>
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/7/7f/RTQPCR.png
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" width = "500"/><figcaption> Figure <b>RTq-PCR of D-NY15 under the control of pGAP promoter</b>The amount of fluorescence provided by the RTq-PCR with the D-NY15 amorces is raising after few cycles (8.32 +/- 0.03) whereas the negative control (pPIC only) start to be amplified at 29 cycles (non specific amplification). This mean that mRNA of DNY15 have been produced. </figcaption></figure>
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We can observed a significant inhibition halo meaning that the AMP have been secreted thanks to the alpha factor.  
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We can observe a significant inhibition halo meaning that the AMP have been secreted thanks to the alpha factor.  
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<p>
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Perspectives : other experiments could be performed with a reporter gene (GFP or RFP for instance) to observe the location of the protein fusionnated with the alpha factor signal in yeast by microscopy.  
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<b>Perspectives:</b> other experiments could be performed with a reporter gene (GFP or RFP for instance) to observe the location of the protein fusionnated with the alpha factor signal in yeast by microscopy.  
  
 
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Revision as of 15:39, 17 October 2017

Alpha-Factor Secretion Signal

The factor secretion signal is a N-terminal secretion signal from S. cerevisiae alpha factor intended to be used to create fusion proteins

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 244
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Team INSA UPS France 2017's usage with Pichia pastoris strain to secret Antimicrobial peptides

We fusionnate the alpha secretion factor with an antimicrobial peptides (AMP). The construction was put under the control of a pGAP promoter.

The construction was cloned in pPICZalpha vector and has been integrated in Pichia pastoris thanks to p(GAP) genomic homology region.

The effect of signal factor was investigated by a toxicity assay with AMP. We intend to see an inhibition effect of the yeast solution if the AMP are secreted i.e. if the alpha secretion factor works properly.

Here is a summary of our result: The engineered yeast were used in a halo assay against V. harveyi as the target of AMPs. A paper soacked with a yeast solution was placed on the plate and V. harveyi growth in the viscinity of the yeast patch was followed:

Figure AMP halo assay Positive control is performed with chloramphenicol (25 g/L), the negative control is performed with empty plasmid integrated in P. pastoris, the assay is performed with plasmid containing BBa_K2278021 integrated in P. pastoris/ The yeast solution was concentrated 10 times
We can observe a significant inhibition halo meaning that the AMP have been secreted thanks to the alpha factor.

Perspectives: other experiments could be performed with a reporter gene (GFP or RFP for instance) to observe the location of the protein fusionnated with the alpha factor signal in yeast by microscopy.