Composite

Part:BBa_K5327035:Design

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


pRS424-ADH1p-GGP1-PYK1t-GPDp-GSTF9-ADH1t-HXT7p-SUR1-HXT7t-ADH1t-UGT-PGK1p


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 1626
    Illegal SpeI site found at 2022
    Illegal PstI site found at 4763
    Illegal PstI site found at 5081
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 1626
    Illegal SpeI site found at 2022
    Illegal PstI site found at 4763
    Illegal PstI site found at 5081
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2662
    Illegal BglII site found at 4895
    Illegal BglII site found at 6449
    Illegal BglII site found at 8935
    Illegal BamHI site found at 1752
    Illegal BamHI site found at 3969
    Illegal BamHI site found at 8830
    Illegal XhoI site found at 6609
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 1626
    Illegal SpeI site found at 2022
    Illegal PstI site found at 4763
    Illegal PstI site found at 5081
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 1626
    Illegal SpeI site found at 2022
    Illegal PstI site found at 4763
    Illegal PstI site found at 5081
    Illegal NgoMIV site found at 180
    Illegal AgeI site found at 1494
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 830
    Illegal BsaI site found at 3770
    Illegal BsaI site found at 6606
    Illegal BsaI.rc site found at 6972
    Illegal SapI site found at 1600
    Illegal SapI site found at 6245


Design Notes

Using CDS sequences from Arabidopsis thaliana (Ath), codon-optimized for S288C, this design employs fundamental molecular biology techniques. The GGP1, GSTF9, SUR1, and UGT genes, which play critical roles in the metabolic pathway, are used as core elements. These genes are combined with ADH1p, PYK1t, GPDp, ADH1t, HXT7p, HXT7t, ADH1t, and PGK1p to create a composite fragment. This combination facilitates the synthesis of the target intermediate, Desulfo-glucosinolate. Subsequent genomic homologous recombination in Saccharomyces cerevisiae S288C ensures the stable expression of this gene fragment, ultimately validating the effective synthesis of sulforaphane intermediates.

Plasmid

Fig 1. The plasmid expression of pRS424-ADH1p-GGP1-PYK1t-GPDp-GSTF9-ADH1t-HXT7p-SUR1-HXT7t-ADH1t-UGT-PGK1p

Source

Arabidopsis thaliana