Difference between revisions of "Part:BBa K1033282"
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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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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.
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[[Image:T--KUAS Korea--amilCP.png|500px|center|]]
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[[File:OD588-time-re-KUAS-Korea.png|500px|center|]]
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[[File:OD588-800-ratio-KUAS-Korea.png|500px|center|]]
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[[File:--File-OD588-800-KUAS-Korea-500px-center--.png|center|]]
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The first photo is a transformation of BBa_K1033282 to DH5alpha. This was then grown in LB medium supplemented with chloramphenicol at 37 degrees Celsius. The first graph shows changes in OD588 over time, the second shows changes in OD588 / OD800 ratio with time, and the third shows OD588 according to OD800.

Latest revision as of 03:29, 28 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.


T--KUAS Korea--amilCP.png


OD588-time-re-KUAS-Korea.png


OD588-800-ratio-KUAS-Korea.png


--File-OD588-800-KUAS-Korea-500px-center--.png


The first photo is a transformation of BBa_K1033282 to DH5alpha. This was then grown in LB medium supplemented with chloramphenicol at 37 degrees Celsius. The first graph shows changes in OD588 over time, the second shows changes in OD588 / OD800 ratio with time, and the third shows OD588 according to OD800.