Composite

Part:BBa_K4012029

Designed by: Jiayi He   Group: iGEM21_AISSU_Union   (2021-10-20)
Revision as of 13:55, 21 October 2021 by Bearbear (Talk | contribs) (→‎Fermentation results by HPLC test)


RtTAL-Pc4CL-GbCHS-MsCHI level2

It is a composite part of naringenin metabolic pathway. pRET2-RtTAL-tSSA1-pPGK1-Pc4CL-tCYC1-pTEF1-GbCHS-tADH1-pPOP6-MsCHI-tTDH1

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 195
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 714
    Illegal BglII site found at 2758
    Illegal BglII site found at 4201
    Illegal BglII site found at 6848
    Illegal BglII site found at 9038
    Illegal XhoI site found at 2844
    Illegal XhoI site found at 8072
    Illegal XhoI site found at 9746
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 50
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3
    Illegal BsaI site found at 2830
    Illegal BsaI site found at 3099
    Illegal BsaI site found at 6137
    Illegal BsaI site found at 8058
    Illegal BsaI site found at 8327
    Illegal BsaI.rc site found at 724
    Illegal BsaI.rc site found at 3080
    Illegal BsaI.rc site found at 4211
    Illegal BsaI.rc site found at 6858
    Illegal BsaI.rc site found at 8308
    Illegal BsaI.rc site found at 9048
    Illegal SapI site found at 7762
    Illegal SapI.rc site found at 7711


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 naringenin, 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.

[[Image: HPLC result.jpeg|thumbnail|750px|center|Figure 2: [2] Fig.1 The results of HPLC of naringenin, taxifolin, eriodictyol, catechin standard and sample from fermentation medium] As shown in Fig.1, 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.

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