Part:BBa_K5327032

ADH1p-BCAT4-CYC1t-CDC19p-MAM1-ADH1t

Function:

This product utilizes the BCAT4 and MAM1 genes to construct a catalytic element aimed at optimizing the biosynthetic pathway of sulforaphane. BCAT4 encodes an aminotransferase and MAM1 encodes a monooxygenase. The process involves the transamination of methionine by BCAT4 and subsequent coupling with acetyl-CoA to produce 2-Alkylmalate, thus facilitating the effective synthesis of intermediates in sulforaphane biosynthesis. This approach integrates vectors into a composite fragment for genomic homologous recombination in the yeast strain (S288C). The gene fragments are transformed into yeast (S288C) and expression is verified to screen for positive clones.

Usage and Biology

Expression diagram:

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

PCR result:

Fig 2. The PCR result of ADH1p-BCAT4-CYC1t-CDC19p-MAM1-ADH1t

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

Source

Arabidopsis thaliana

Sequence and Features

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