Composite

Part:BBa_K5175038:Design

Designed by: Yuhan Zou   Group: iGEM24_HUST-China   (2024-10-01)


T7 promoter - tphA2-tphA3-tphB -tphA1-T7 terminator


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 912
    Illegal PstI site found at 949
    Illegal PstI site found at 982
    Illegal PstI site found at 2938
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3813
    Illegal NheI site found at 3878
    Illegal PstI site found at 949
    Illegal PstI site found at 982
    Illegal PstI site found at 2938
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 426
    Illegal BglII site found at 2027
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 912
    Illegal PstI site found at 949
    Illegal PstI site found at 982
    Illegal PstI site found at 2938
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 912
    Illegal PstI site found at 949
    Illegal PstI site found at 982
    Illegal PstI site found at 2938
    Illegal NgoMIV site found at 477
    Illegal NgoMIV site found at 1516
    Illegal NgoMIV site found at 2074
    Illegal NgoMIV site found at 2749
    Illegal NgoMIV site found at 3253
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

It is a composite component consisting of the T7 promoter, T7 terminator, target genes tphA2, tphA3, tphB, tphA1. It is responsible for converting TPA to 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD), oxidising the diol moiety (two hydroxyl groups) of DCD to a keto group to result in the production of PCA. TphA2, TphA3 constitute the large and small subunits of the TPADO oxidase component responsible for binding to the TPA substrate and catalysing the oxygenation reaction in the active site. TphA1 contains a [2Fe -2S] iron-sulfur cluster and a flavin adenine dinucleotide (FAD) binding site that transfers electrons from an electron donor (e.g., NADPH) to the oxidised component of TPADO. TphB is a dehydrogenase that oxidises the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting in the production of PCA.



Source

tphA2, tphA3, tphB, tphA1 are from Comamonas sp.E6.

References

[1] NIKEL P I, DE LORENZO V. Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism [J]. Metab Eng, 2018, 50: 142-55.

[2] HARWOOD C S, PARALES R E. THE β-KETOADIPATE PATHWAY AND THE BIOLOGY OF SELF-IDENTITY [J]. Annual Review of Microbiology, 1996, 50(Volume 50, 1996): 553-90.

[3] SASOH M, MASAI E, ISHIBASHI S, et al. Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6 [J]. Applied and Environmental Microbiology, 2006, 72(3): 1825-32.