Difference between revisions of "Part:BBa K2950000"
 
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<partinfo>BBa_K2950000 short</partinfo>
 
<partinfo>BBa_K2950000 short</partinfo>
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<img src="https://2019.igem.org/wiki/images/6/6f/T--BNDS_China--CA_biosynthesis_pathway.png" width=600px></br>
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<p style="width:600px; margin-left:150px; margin-bottom:60px;
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"><em><strong>FIGURE 1</strong>the biosynthesis pathway of carminic acid</em> </p>
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In our study, we aim to achieve carminic acid yield by S. cerevisiae, so we should firstly realize the production of the flavokermesic acid which is an intermediate between acetyl-CoA or malonyl-CoA and carminic acid in the biosynthesis pathway.  
 
In our study, we aim to achieve carminic acid yield by S. cerevisiae, so we should firstly realize the production of the flavokermesic acid which is an intermediate between acetyl-CoA or malonyl-CoA and carminic acid in the biosynthesis pathway.  
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At the beginning, we transformed a plasmid with pYES as the copy vector to keep high copy number, the Aloe arborescens octaketide synthase (OKS) to synthesize non-reduced linear octaketide, and the ZhuI cyclase to convert non-reduced linear octaketide to flavokermesic acid.  
 
At the beginning, we transformed a plasmid with pYES as the copy vector to keep high copy number, the Aloe arborescens octaketide synthase (OKS) to synthesize non-reduced linear octaketide, and the ZhuI cyclase to convert non-reduced linear octaketide to flavokermesic acid.  
  
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After the transformation and the verification of colony PCR, we got a strain with the expression of OKS and ZhuI theoretically. Because the High performance liquid chromatography (HPLC) is not available, we can only verify the result qualitatively, and the way is that flavokermesic acid shows a dark red color so that we can observe whether the yeast colony on the culture plate shows red color. Here are two figures showing the phenomenon.  
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After the transformation and the verification of colony PCR, we got a strain with the expression of OKS and ZhuI theoretically. Because the High performance liquid chromatography (HPLC) and the standard sample FK are both unavailable, we can only verify the result qualitatively, and the way is that flavokermesic acid shows a dark red color so that we can observe whether the yeast colony on the culture plate shows red color. Here are two figures showing the phenomenon.  
  
 
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<img src="https://2019.igem.org/wiki/images/3/3f/T--BNDS_China--FK_solid.jpeg" width=600px>
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<img src="https://static.igem.org/mediawiki/parts/1/19/T--BNDS_China--OKS_ZhuI.png" width=600px></br>
 
<p style="width:600px; margin-left:150px; margin-bottom:60px;
 
<p style="width:600px; margin-left:150px; margin-bottom:60px;
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"><em><strong>FIGURE 1</strong>The yeast colony on the solid culture medium</em> </p>
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"><em><strong>FIGURE 2</strong>The agarose gel electrophoresis about OKS-ZhuI</em> </p>
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<img src="https://2019.igem.org/wiki/images/b/b2/T--BNDS_China--FK_liquid.jpg" width=600px>
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<img src="https://2019.igem.org/wiki/images/3/3f/T--BNDS_China--FK_solid.jpeg" width=600px></br>
 
<p style="width:600px; margin-left:150px; margin-bottom:60px;
 
<p style="width:600px; margin-left:150px; margin-bottom:60px;
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"><em><strong>FIGURE 2</strong>The yeast precipitation in the liquid culture medium</em> </p>
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"><em><strong>FIGURE 3</strong>The yeast colony on the solid culture medium</em> </p>
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<img src="https://2019.igem.org/wiki/images/b/b2/T--BNDS_China--FK_liquid.jpg" width=600px></br>
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<p style="width:600px; margin-left:150px; margin-bottom:60px;
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text-align:center
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"><em><strong>FIGURE 4</strong>The yeast precipitation in the liquid culture medium</em> </p>
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Latest revision as of 02:28, 22 October 2019


pYES-GAL1 promoter-OKS-TDH1 terminator-TEF1 promoter-ZhuI-CYC1 terminator



FIGURE 1the biosynthesis pathway of carminic acid


In our study, we aim to achieve carminic acid yield by S. cerevisiae, so we should firstly realize the production of the flavokermesic acid which is an intermediate between acetyl-CoA or malonyl-CoA and carminic acid in the biosynthesis pathway.

At the beginning, we transformed a plasmid with pYES as the copy vector to keep high copy number, the Aloe arborescens octaketide synthase (OKS) to synthesize non-reduced linear octaketide, and the ZhuI cyclase to convert non-reduced linear octaketide to flavokermesic acid.

After the transformation and the verification of colony PCR, we got a strain with the expression of OKS and ZhuI theoretically. Because the High performance liquid chromatography (HPLC) and the standard sample FK are both unavailable, we can only verify the result qualitatively, and the way is that flavokermesic acid shows a dark red color so that we can observe whether the yeast colony on the culture plate shows red color. Here are two figures showing the phenomenon.


FIGURE 2The agarose gel electrophoresis about OKS-ZhuI


FIGURE 3The yeast colony on the solid culture medium



FIGURE 4The yeast precipitation in the liquid culture medium

According to figure 1 and figure 2, the yeast colony and its precipitation in the liquid medium both show a significant dark red color, which means that we successfully characterize this pGAL1-OKS-tTDH1-pTEF1-ZhuI-tCYC1 composite part qualitatively by showing its red color.

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 1769
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 150
  • 1000
    COMPATIBLE WITH RFC[1000]