Composite

Part:BBa_K5327033

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


p426-TEF1p-LSU-ADH1t-ADH1p-IPMI2-PYK1t-ENO2p-BCAT3-HXT7t-GPDp-IPMDH1-CYC1t

Function:

This product constructs a catalytic element using IPMI, IPMDH1, and BCAT3, forming one of the steps in the sulforaphane biosynthesis pathway. Together with the (BCAT4-MAM1) plasmid BBa_K5327032, it completes the first part of this pathway—side-chain elongation.

The process involves the isomerization of 2-Alkylmalate, oxidative decarboxylation, and transamination to ultimately synthesize the intermediate Dihomomethionine. This enables the effective synthesis of sulforaphane intermediates. The process integrates three vectors into a composite fragment, facilitating 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 p426-TEF1p-LSU-ADH1t-ADH1p-IPMI2-PYK1t-ENO2p-BCAT3-HXT7t-GPDp-IPMDH1-CYC1t

PCR result:

Fig 2. The PCR result of p426-TEF1p-LSU-ADH1t-ADH1p-IPMI2-PYK1t-ENO2p-BCAT3-HXT7t-GPDp-IPMDH1-CYC1t

Design Notes

The design utilizes CDS sequences from Arabidopsis thaliana (Ath) and is codon-optimized for S288C. Core elements IPMI, IPMDH1, and BCAT3 are employed, which play crucial roles in the metabolic pathway. These genes are combined with TEF1p, ADH1t, ADH1p, PYK1t, ENO2p, HXT7t, GPDp, and CYC1t to create a composite fragment. This combination allows for the synthesis of the target intermediate dihomomethionine. Subsequently, 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 3. The plasmid expression of p426-TEF1p-LSU-ADH1t-ADH1p-IPMI2-PYK1t-ENO2p-BCAT3-HXT7t-GPDp-IPMDH1-CYC1t

Source

Arabidopsis thaliana



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1745
    Illegal BglII site found at 7892
    Illegal BglII site found at 8086
    Illegal BamHI site found at 819
    Illegal XhoI site found at 4353
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2517
    Illegal AgeI site found at 4449
    Illegal AgeI site found at 4490
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 620
    Illegal BsaI site found at 3167
    Illegal BsaI site found at 6478
    Illegal BsaI.rc site found at 205
    Illegal BsaI.rc site found at 7859
    Illegal BsaI.rc site found at 7916


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Parameters
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