Difference between revisions of "Part:BBa K431009"

 
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==Team INSA UPS France 2017's usage with <i>Pichia pastoris</i> strain to produce Antimicrobial peptides==
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p(GAP)
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We use pGAP as a constitutive to produce RFP and to secrete antimicrobial peptides against cholera. As pGAP was a crucial part of the system we tried to characterize it in Pichia pastoris strain.
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<p>The construction has been integrated in Pichia pastoris thanks to p(GAP) genomic homology region.
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<p>The promoter effect of p(GAP) was investigated with a RFP reporter gene by fluorimeter and by performing a RT-qPCR with the RNAm of the peptides placed downstream the promoter.
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Here is a summary of our result : 
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RT-qPCR (from BBa_K2278021)
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figure coming soon
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<figure><p style="text-align:center;"><img src="" width = "500"/><figcaption> Figure  <b>RTq-PCR of D-NY15 </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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Revision as of 15:17, 17 October 2017


glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP)

This promoter is responsible for the transcription of glyceraldehyde 3-phosphate dehydrogenase in Pichia pastoris. Because this is a key enzyme for glycolysis, it is a strong constitutive promoter. This promoter is one of the most common alternatives to pAOX1 for heterologous protein production in P. pastoris. It is advantageous because its use obviates the hazards and expenses involved with using methanol and still provides high yields of protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 245


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

p(GAP) We use pGAP as a constitutive to produce RFP and to secrete antimicrobial peptides against cholera. As pGAP was a crucial part of the system we tried to characterize it in Pichia pastoris strain.

The construction has been integrated in Pichia pastoris thanks to p(GAP) genomic homology region.

The promoter effect of p(GAP) was investigated with a RFP reporter gene by fluorimeter and by performing a RT-qPCR with the RNAm of the peptides placed downstream the promoter. Here is a summary of our result : RT-qPCR (from BBa_K2278021) figure coming soon

Figure RTq-PCR of D-NY15 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.