Generator

Part:BBa_K4462111

Designed by: Kredai Raaman   Group: iGEM22_IISc-Bengaluru   (2022-09-30)


pTOD

Construct for constitutive expression of the Toluene dioxygenase enzyme system


Usage and Biology

Toluene dioxygenase is present naturally in strains of Pseudomonas putida wherein it converts a double bond in toluene to a vicinal diol. The fundamental motivation for using this enzyme lies in the oxidation of double bonds something which exists in many halocarbons and also exists in products reduced by Cytochrome P450cam. It is a heterotrimeric protein which has 3 major components, an NADH reductase, coded by TodA gene, a Ferredoxin coded by TodB and an oxygenase. The oxygenase has an alpha and a beta subunit coded by TodC1 and TodC2 genes respectively. It has a pH optima of 6-7.5 and a temperature optima of 28 degree celsius.

Under aerobic conditions it has shown to convert halocarbons with at least one double to vicinal diols and then further converts the remaining halogens on the halogenated vicinal diols to OH groups. Conversion from a double bonded structure to a vicinal diol is trivial and occurs naturally with this enzyme. The second conversion is a hypothetial route for degradation of single carbon halocarbons in our system. The fundamental role of toluene dioxygenase lies in degrading substrates containing one carbon, degrading substrates containing a double bond and degrading the product formed from cytochrome P450 cam degradation under anaerobic conditions.



The plasmid pTOD was designed to expresses the four-component Toluene dioxygenase enzyme system as a reconstituted operon under the control of the strong constitutive EM7 promoter (pTOD6). The operon as designed consists of the TodC1, TodC2, TodA, and TodB genes and was supposed to be inserted into pSEVA2213 (medium-copy, kanamycin-resistance, EM7 promoter).




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 2570
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3578
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 450
    Illegal NgoMIV site found at 648
    Illegal NgoMIV site found at 1498
    Illegal NgoMIV site found at 3256
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3622


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Parameters
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