Composite

Part:BBa_K4012030

Designed by: Jiayi He   Group: iGEM21_AISSU_Union   (2021-10-20)


CrCPR-CsF3H-GhF3'H-AaDFR-DuLAR level2

It is a composite part of catechin metabolic pathway. pGAL1-CrCPR-tTDH1-pTEF1-CsF3H-tENO2-pPGK1-GHF3'H-tADH1-pTDH3-AaDFR-tENO1-pPOP6-DuLAR-tSSA1

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 547
    Illegal BglII site found at 781
    Illegal BglII site found at 1516
    Illegal BglII site found at 1564
    Illegal BglII site found at 3682
    Illegal BglII site found at 3865
    Illegal BglII site found at 6171
    Illegal BamHI site found at 7394
    Illegal XhoI site found at 2731
    Illegal XhoI site found at 4828
    Illegal XhoI site found at 7758
    Illegal XhoI site found at 9765
    Illegal XhoI site found at 11914
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 163
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3
    Illegal BsaI site found at 2971
    Illegal BsaI site found at 5069
    Illegal BsaI site found at 7744
    Illegal BsaI site found at 10006
    Illegal BsaI site found at 11900
    Illegal BsaI.rc site found at 557
    Illegal BsaI.rc site found at 3692
    Illegal BsaI.rc site found at 6181
    Illegal BsaI.rc site found at 7994
    Illegal BsaI.rc site found at 10727
    Illegal BsaI.rc site found at 12150
    Illegal SapI site found at 9463


Level2 plasmid assembly

Figure 1: [1] A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway

Assembly of synthesis pathway of catechin, shown by Fig.1.

Fermentation results by HPLC test

During the HPLC testing, we purchased final and intermediate metabolism products standard, naringenin, taxifolin, eriodictyol and catechin, and used them in the subsequent HPLC tests.

Figure 2: [2] Fig.2 The results of HPLC of naringenin, taxifolin, eriodictyol, catechin standard and sample from fermentation medium

As shown in Fig.2, since we could not buy leucoanthocyanidins (LCD), we only did HPLC of the four intermediates: Catechin, Eriodictyol, Taxifolin and Naringenin. Catechin and other intermediate metabolites were confirmed by comparing the samples with the standard. This demonstrate that the part can produce narigenin through fermentation.

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