Difference between revisions of "Part:BBa K1033282"
(→‎Team INSA-UPS France 2017: demonstration of pGAP validity in Pichia pastoris)
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<partinfo>BBa_K1033282 parameters</partinfo>
 
<partinfo>BBa_K1033282 parameters</partinfo>
 
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==Team INSA-UPS France 2017: demonstration of pGAP validity in <i>Pichia pastoris</i>==
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==Team KUAS_Korea 2017: characterization==
  
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We tested the functionality of pGAP as a constitutive promoter in the yeast <i>Pichia pastoris</i> background.  
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We tested the functionality of K1033222 as a constitutive promoter and expression of amilCP chromoprotein in the <i>Escherichia coli</i> DH5alpha.  
  
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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 <partinfo>BBa_K2278021</partinfo> (D-NY15 antimicrobial peptide encoding gene for our project).  
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DH5alpha containing the control(pSB1C3) and BBa_K1033282, respectively, were cultured in LB medium containing chloramphenicol.
  
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Absorbances were measured every 30 minutes using BioRad SmartSpecPlus at 588 nm and 800 nm.
  
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[[Image:T--INSA-UPS_France--RTQPCRdekalitaäy.png|800px|thumb|center|<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).]]
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[[Image:T--KUAS Korea--amilCP.png|800px|thumb|center|<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).]]
  
 
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 as a promoter. This promoter is therefore very efficient in the <i>Pichia pastoris</i> background.
 
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 as a promoter. This promoter is therefore very efficient in the <i>Pichia pastoris</i> background.

Revision as of 11:13, 27 October 2017

amilCP blue chromoprotein with strong promoter

This is a blue/purple chromoprotein (amilCP: BBa_K592009) with the RBS B0034 (BBa_B0034) and a strong promoter (CP29: BBa_K1033222). The construct has been shown to work in E. coli and should work in Lactobacillus.

(Construct name: pSB1C3-CP29-B0034-amilCP)

See http://2013.igem.org/Team:Uppsala/reporter-genes for more information.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Team KUAS_Korea 2017: characterization

We tested the functionality of K1033222 as a constitutive promoter and expression of amilCP chromoprotein in the Escherichia coli DH5alpha.

DH5alpha containing the control(pSB1C3) and BBa_K1033282, respectively, were cultured in LB medium containing chloramphenicol.

Absorbances were measured every 30 minutes using BioRad SmartSpecPlus at 588 nm and 800 nm.


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 as a promoter. This promoter is therefore very efficient in the Pichia pastoris background.