Difference between revisions of "Part:BBa K1800001"

(→‎Team INSA-UPS France 2017: usage of BBa_K1800001 in Pichia pastoris strain to secrete antimicrobial peptides)
 
(5 intermediate revisions by the same user not shown)
Line 19: Line 19:
  
 
==Team INSA-UPS France 2017: usage of BBa_K1800001 in <i>Pichia pastoris</i> strain to secrete antimicrobial peptides==
 
==Team INSA-UPS France 2017: usage of BBa_K1800001 in <i>Pichia pastoris</i> strain to secrete antimicrobial peptides==
−
<html>
 
  
  
−
We fusionnate the alpha secretion factor with an antimicrobial peptide (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 (<partinfo>BBa_K431009</partinfo>).
−
<p>The construction was cloned in pPICZalpha vector and has been integrated in <i>Pichia pastoris</i> thanks to pGAP genomic homology region.  
+
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.  
  
−
<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:
+
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:
−
</html>
+
−
[[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 BBa_K2278021 driving D-NY15 secretion integrated in <i>P. pastoris</i>. ]]
+
  
−
<html>
+
[[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>. ]]
  
−
We can observe a significant inhibition halo meaning that the AMP have been secreted thanks to the alpha factor.
 
−
<p>
 
  
−
 
+
We can observe a significant inhibition halo meaning that the AMP have been secreted thanks to the alpha factor.
−
</html>
+

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.