Signalling

Part:BBa_K1800001

Designed by: Laura Lee Irvin   Group: iGEM15_Georgia_State   (2015-08-28)
Revision as of 20:34, 23 October 2017 by Pzanoni (Talk | contribs) (→‎Team INSA-UPS France 2017: usage of BBa_K1800001 in Pichia pastoris strain to secrete antimicrobial peptides)

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_K431007BBa_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.

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Categories
Parameters
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