Difference between revisions of "Part:BBa K1800001"
 
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==Team INSA UPS France 2017's usage with <i>Pichia pastoris</i> strain to secret Antimicrobial peptides==
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==Team INSA-UPS France 2017: usage of BBa_K1800001 in <i>Pichia pastoris</i> strain to secrete antimicrobial peptides==
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We fusionnate the alpha secretion factor with an antimicrobial peptides (AMP). The construction was put under the control of a pGAP promoter.
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We fusionnate the alpha secretion factor with an antimicrobial peptide (AMP). The construction was put under the control of a pGAP promoter (<partinfo>BBa_K431009</partinfo>).
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<p>The construction was cloned in pPICZalpha vector and has been integrated in Pichia pastoris thanks to p(GAP) genomic homology region.  
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The construction was cloned in pPICZalpha vector and has been integrated in <i>Pichia pastoris</i> thanks to pAOXI (<partinfo>BBa_K431007</partinfo>) genomic homology region.  
  
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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.
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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:
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Here is a summary of our result :  
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[[Image:T--INSA-UPS_France--HalosPichia.png|800px|thumb|center|<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 ( <partinfo>BBa_K2278021</partinfo>) driving D-NY15 secretion integrated in <i>P. pastoris</i>. ]]
  
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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/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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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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Latest revision as of 20:50, 23 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 (BBa_K431009). The construction was cloned in pPICZalpha vector and has been integrated in Pichia pastoris thanks to pAOXI (BBa_K431007) 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.