Difference between revisions of "Part:BBa K1800001"

(→‎Team INSA UPS France 2017's usage with Pichia pastoris strain to secret Antimicrobial peptides)
Line 18: Line 18:
  
  
−
==Team INSA UPS France 2017's usage with <i>Pichia pastoris</i> strain to secret Antimicrobial peptides==
+
==Team INSA-UPS France 2017: usage of BBa_K1800001 in <i>Pichia pastoris</i> strain to secrete antimicrobial peptides==
 
<html>
 
<html>
  
  
−
We fusionnate the alpha secretion factor with an antimicrobial peptides (AMP). The construction was put under the control of a pGAP promoter.  
+
We fusionnate the alpha secretion factor with an antimicrobial peptide (AMP). The construction was put under the control of a pGAP promoter.  
−
<p>The construction was cloned in pPICZalpha vector and has been integrated in Pichia pastoris thanks to p(GAP) genomic homology region.  
+
<p>The construction was cloned in pPICZalpha vector and has been integrated in <i>Pichia pastoris</i> thanks to pGAP genomic homology region.  
  
−
<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 functionality of the signal factor was investigated by demonstrating that AMP are present in the supernatant through a toxicity assay. The engineered yeast was used in a halo assay against <i>V. harveyi</i> as the target of AMPs. A paper soacked with a yeast supernantant solution was placed on the plate and <i>V. harveyi</i> growth in the viscinity of the yeast patch was followed:
−
<p> 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><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/5/5c/Halos_pichia.png" width = "500"/><figcaption> <b>AMP halo assay</b> Positive control was performed with chloramphenicol (25 g/L), the negative control was performed with the empty plasmid integrated in <i>P. pastoris</i>, the assay was performed using the plasmid containing BBa_K2278021 driving D-NY15 secretion integrated in <i>P. pastoris</i>/ </figcaption></figure>
−
 
+
−
<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>
+
  
 
We can observe a significant inhibition halo meaning that the AMP have been secreted thanks to the alpha factor.  
 
We can observe a significant inhibition halo meaning that the AMP have been secreted thanks to the alpha factor.  
 
<p>
 
<p>
−
<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.
+
 
  
 
</html>
 
</html>

Revision as of 09:33, 19 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: usage of BBa_K1800001 in Pichia pastoris strain to secrete antimicrobial peptides

We fusionnate the alpha secretion factor with an antimicrobial peptide (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 pGAP genomic homology region.

The functionality of the signal factor was investigated by demonstrating that AMP are present in the supernatant through a toxicity assay. The engineered yeast was used in a halo assay against V. harveyi as the target of AMPs. A paper soacked with a yeast supernantant solution was placed on the plate and V. harveyi growth in the viscinity of the yeast patch was followed:

AMP halo assay Positive control was performed with chloramphenicol (25 g/L), the negative control was performed with the empty plasmid integrated in P. pastoris, the assay was performed using the plasmid containing BBa_K2278021 driving D-NY15 secretion integrated in P. pastoris/
We can observe a significant inhibition halo meaning that the AMP have been secreted thanks to the alpha factor.