Composite

Part:BBa_K5327036

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


PGI1p-SOT18-HXT7t-ADH1p-ATR2-TDH2t-GPDp-FMO-PYK1t

Function:

This product utilizes SOT18, ATR2, and FMO genes to construct a catalytic element, representing one of the steps in sulforaphane production. Together with the plasmid BBa_K5327035, it completes the third part of the pathway—synthesis of core glucosinolates and modification of amino acid side chains—successfully synthesizing the precursor of the product, sulforaphane.

The process involves the sulfation and sulfoxidation of Desulfo-glucosinolate to synthesize the precursor compound Glucoraphanin. This is achieved by combining three vectors to form a composite fragment, facilitating genomic homologous recombination in the yeast strain S288C. The constructed gene fragment is transformed into yeast (S288C) and expressed, followed by screening for positive clones.

Usage and Biology

Expression diagram:

Fig 1. The expression diagram of PGI1p-SOT18-HXT7t-ADH1p-ATR2-TDH2t-GPDp-FMO-PYK1t

Design Notes

Utilizing CDS sequences from Arabidopsis thaliana (ath), optimized for S288C codon usage, this design is based on fundamental molecular biology techniques. The genes SOT18, ATR2, and FMO are employed as core elements, playing critical roles in the metabolic pathway. These genes are combined with PGI1p, HXT7t, ADH1p, TDH2t, GPDp, and PYK1t to create a composite BBa fragment. This combination facilitates the synthesis of the sulforaphane precursor Glucoraphanin. Subsequent genomic homologous recombination in Saccharomyces cerevisiae S288C ensures stable expression of this gene fragment, ultimately validating the effective synthesis of sulforaphane intermediates.

Plasmid

Fig 2. The plasmid expression of PGI1p-SOT18-HXT7t-ADH1p-ATR2-TDH2t-GPDp-FMO-PYK1t

Source

Arabidopsis thaliana




Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 5501
    Illegal EcoRI site found at 6172
    Illegal EcoRI site found at 6271
    Illegal XbaI site found at 5748
    Illegal SpeI site found at 4520
    Illegal PstI site found at 6082
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 5501
    Illegal EcoRI site found at 6172
    Illegal EcoRI site found at 6271
    Illegal NheI site found at 5999
    Illegal SpeI site found at 4520
    Illegal PstI site found at 6082
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 5501
    Illegal EcoRI site found at 6172
    Illegal EcoRI site found at 6271
    Illegal BamHI site found at 5841
    Illegal XhoI site found at 5348
    Illegal XhoI site found at 5357
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 5501
    Illegal EcoRI site found at 6172
    Illegal EcoRI site found at 6271
    Illegal XbaI site found at 5748
    Illegal SpeI site found at 4520
    Illegal PstI site found at 6082
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 5501
    Illegal EcoRI site found at 6172
    Illegal EcoRI site found at 6271
    Illegal XbaI site found at 5748
    Illegal SpeI site found at 4520
    Illegal PstI site found at 6082
    Illegal NgoMIV site found at 2342
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2992
    Illegal BsaI site found at 3896
    Illegal BsaI site found at 5112
    Illegal BsaI site found at 5187
    Illegal BsaI site found at 6305
    Illegal BsaI.rc site found at 657
    Illegal BsaI.rc site found at 743
    Illegal BsaI.rc site found at 4085
    Illegal SapI.rc site found at 4204


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Parameters
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