Composite

Part:BBa_K5327034

Designed by: Fangxian Chen   Group: iGEM24_BUCT   (2024-09-25)


CDC19p-CYP79F1-PYK1t-PGI1p-CYP83A1-HXT7t

Function:

This product employs CYP79F1 and CYP83A1 genes to construct a catalytic element, aiming to further optimize the biosynthetic pathway of sulforaphane. CYP79F1 catalyzes the conversion of Dihomomethionine (DHM) to aldoxime, while CYP83A1 subsequently oxidizes aldoxime to an aci-nitro compound, achieving the oxidation transformation of DHM. This process represents a critical step in the sulforaphane biosynthesis pathway. The constructed gene fragments are integrated into the genome of the yeast strain S288C through genomic homologous recombination. The transformed yeast cells are then subjected to expression validation to screen for positive clones.

Usage and Biology

Expression diagram:

Fig 1. The expression diagram of CDC19p-CYP79F1-PYK1t-PGI1p-CYP83A1-HXT7t

PCR result:

Fig 2. The PCR result of CDC19p-CYP79F1-PYK1t-PGI1p-CYP83A1-HXT7t


Design Notes

Utilizing CDS sequences from Arabidopsis thaliana (Ath), codon-optimized for S288C, this design employs fundamental molecular biology techniques. The CYP79F1 and CYP83A1 genes, which are pivotal in the metabolic pathway, are used as core elements. These genes are combined with CDC19p, PYK1t, PGI1p, and HXT7t to form a composite fragment, enabling the synthesis of the target intermediate, the aci-nitro compound. CYP79F1 and CYP83A1 are key cytochrome P450 enzymes that play essential roles in oxidative catalysis within the sulforaphane synthesis pathway, providing precursors for subsequent reactions.

Plasmid

Fig 3. The plasmid expression of CDC19p-CYP79F1-PYK1t-PGI1p-CYP83A1-HXT7t

Source

Arabidopsis thaliana




Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2172
    Illegal SpeI site found at 1051
    Illegal PstI site found at 4643
    Illegal PstI site found at 5522
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2172
    Illegal NheI site found at 3978
    Illegal SpeI site found at 1051
    Illegal PstI site found at 4643
    Illegal PstI site found at 5522
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2172
    Illegal BglII site found at 4373
    Illegal XhoI site found at 1576
    Illegal XhoI site found at 2525
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2172
    Illegal SpeI site found at 1051
    Illegal PstI site found at 4643
    Illegal PstI site found at 5522
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 525
    Illegal EcoRI site found at 2172
    Illegal SpeI site found at 1051
    Illegal PstI site found at 4643
    Illegal PstI site found at 5522
    Illegal NgoMIV site found at 1268
    Illegal AgeI site found at 2408
  • 1000
    COMPATIBLE WITH RFC[1000]


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