Difference between revisions of "Part:BBa K431009"

(→‎Team INSA UPS France 2017's usage with Pichia pastoris strain to produce Antimicrobial peptides)
(→‎Team INSA UPS France 2017's usage with Pichia pastoris strain to produce Antimicrobial peptides)
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<partinfo>BBa_K431009 parameters</partinfo>
 
<partinfo>BBa_K431009 parameters</partinfo>
 
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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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==Team INSA-UPS France 2017: demonstration of pGAP validity in <i>Pichia pastoris</i>==
 
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<html>
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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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We tested the functionality of pGAP BBa_K431009 as a constitutive promoter in the yeast <i>Pichia pastoris</i> background.  
  
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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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<p>The biobrick was cloned in pPICZalpha vector and was integrated in <i>Pichia pastoris</i> at the pGAP genomic locus. The reporter gene here was D-NY15 (an antimicrobial peptide encoding gene for our project).  
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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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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/d/d5/RTQPCRdekalitaäy.png" width = "500"/><figcaption> Figure 6 :<b>RTq-PCR of D-NY15 driven by BBa_K431009 promoter. </b>RNA were issued from <i>Pichia pastoris</i> strains with or without the D-NY15 encoding gene. Total RNAs were extracted from transformants and reverse transcriptions were performed using Superscript II reverse transcriptase (Invitrogen). Resulting D-NY15 cDNA were then amplified by quantitative PCR. The curves correspond to <i>P. pastoris</i> with integrated pPICZalpha-DNY15 (A1, A2, A3), water control (B1 & B2), and <i>P. pastoris</i> with integrated pPICZalpha (C1, C2, C3).</figcaption></figure>
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RT-qPCR (from BBa_K2278021)
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The amount of fluorescence provided by the qRT-PCR with the D-NY15 primers rose after 8 cycles whereas the negative control (pPICZalpha only) started to be amplified at over 29 cycles (non specific amplification). This means that the D-NY15 encoding gene is well expressed in <i>Pichia pastoris</i> with pGAP BBa_K431009 as a promoter. This promoter is therefore very efficient in the <i>Pichia pastoris</i> background.
  
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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>
 
  
  

Revision as of 09:24, 19 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: demonstration of pGAP validity in Pichia pastoris

We tested the functionality of pGAP BBa_K431009 as a constitutive promoter in the yeast Pichia pastoris background.

The biobrick was cloned in pPICZalpha vector and was integrated in Pichia pastoris at the pGAP genomic locus. The reporter gene here was D-NY15 (an antimicrobial peptide encoding gene for our project).

Figure 6 :RTq-PCR of D-NY15 driven by BBa_K431009 promoter. RNA were issued from Pichia pastoris strains with or without the D-NY15 encoding gene. Total RNAs were extracted from transformants and reverse transcriptions were performed using Superscript II reverse transcriptase (Invitrogen). Resulting D-NY15 cDNA were then amplified by quantitative PCR. The curves correspond to P. pastoris with integrated pPICZalpha-DNY15 (A1, A2, A3), water control (B1 & B2), and P. pastoris with integrated pPICZalpha (C1, C2, C3).
The amount of fluorescence provided by the qRT-PCR with the D-NY15 primers rose after 8 cycles whereas the negative control (pPICZalpha only) started to be amplified at over 29 cycles (non specific amplification). This means that the D-NY15 encoding gene is well expressed in Pichia pastoris with pGAP BBa_K431009 as a promoter. This promoter is therefore very efficient in the Pichia pastoris background.