Composite

Part:BBa_K5327032:Design

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


ADH1p-BCAT4-CYC1t-CDC19p-MAM1-ADH1t


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3392
    Illegal SpeI site found at 2358
    Illegal SpeI site found at 4780
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3392
    Illegal SpeI site found at 2358
    Illegal SpeI site found at 4780
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3392
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3392
    Illegal SpeI site found at 2358
    Illegal SpeI site found at 4780
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3392
    Illegal SpeI site found at 2358
    Illegal SpeI site found at 4780
    Illegal NgoMIV site found at 180
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 830
    Illegal BsaI site found at 1734
    Illegal BsaI site found at 4156
    Illegal BsaI.rc site found at 1923
    Illegal BsaI.rc site found at 4345
    Illegal SapI.rc site found at 2042
    Illegal SapI.rc site found at 4464


Design Notes

The CDS sequences from Arabidopsis thaliana (ath) are used and codon-optimized for S288C. The BCAT4 and MAM1 genes are combined with the ADH1p, CYC1t, CDC19p, and ADH1t elements to create a composite vector. Based on fundamental molecular biology techniques, a specific gene expression vector is constructed to achieve the synthesis of the target compound. Finally, genomic homologous recombination is performed in Saccharomyces cerevisiae S288C to integrate the constructed gene fragments into the yeast genome, ensuring stable expression. Expression verification in yeast will be used to select positive clones that successfully integrate and express the target genes, ultimately leading to the biosynthesis of sulforaphane.

Plasmid

Fig 1. The plasmid expression of ADH1p-BCAT4-CYC1t-CDC19p-MAM1-ADH1t

Source

Arabidopsis thaliana