Difference between revisions of "Part:BBa K5175009"

 
 
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TPA 1,2-dioxygenase (TPADO) is a two-component oxygenase consisting of three parts, TphA1, TphA2, and TphA3, which together enable TPADO to effectively catalyze the oxidative reaction of TPA, converting TPA to the intermediate product 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD). TphB is a dehydrogenase that oxidizes the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting 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 catalyzing the oxygenation reaction in the active site.TphA2 contains the active site in direct contact with the substrate, TPA, and contains the Cys-X1-His X17-Cys-X2-His pattern, binds to Rieske-type [2Fe-2S] iron-sulfur clusters and participates in electron transfer, which is a key part of the catalytic reaction of dioxygenases , TphA3 participates in the construction of the substrate channel or the appropriate positioning of the active site, and assists TphA2 in completing the oxidation reaction; TphA1 does not directly participate in the oxygenation reaction, but it 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 oxidized component of TPADO.
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TphB is a dehydrogenase that oxidizes the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting in the production of PCA.
  
 
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<h1>Usage and Biology</h1>
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TPA 1,2-dioxygenase (TPADO) is a two-component oxygenase consisting of three parts, TphA1, TphA2, and TphA3, which together enable TPADO to effectively catalyze the oxidative reaction of TPA, converting TPA to the intermediate product 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD). TphB is a dehydrogenase that oxidizes the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting 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 catalyzing the oxygenation reaction in the active site.TphA2 contains the active site in direct contact with the substrate, TPA, and contains the Cys-X1-His X17-Cys-X2-His pattern, binds to Rieske-type [2Fe-2S] iron-sulfur clusters and participates in electron transfer, which is a key part of the catalytic reaction of dioxygenases , TphA3 participates in the construction of the substrate channel or the appropriate positioning of the active site, and assists TphA2 in completing the oxidation reaction; TphA1 does not directly participate in the oxygenation reaction, but it 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 oxidized component of TPADO.
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<figcaption>Fig 1. tph gene cluster core mechanism of converting TPA to PCA</figcaption>
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<h1>Molecular cloning</h1>
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Initially, we transformed the company-synthesized plasmids containing designed sequences into <i>E. coli</i> DH5α for amplification, allowing us to obtain a sufficient quantity of plasmid DNA for subsequent experiments. Following this, colony PCR was performed to confirm successful transformation, and the required plasmids were subsequently extracted for further experimentation.Subsequently, we employed PCR to obtain the target fragments, which were then integrated into the requisite plasmids for our study.<br><br>We constructed three plasmids for <i>P. putida</i> KT2440: pTerephthalate-A, pTerephthalate-B, and pRhamnolipid. We verified the size of each plasmid as well as all the fragments involved in constructing the plasmids .
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<figcaption>Fig.2  The bands of pTerephthalate-A and pTerephthalate-B from PCR<br><br>The bands of pTerephthalate-A（~3000bp）from PCR are identical to the theoretical lengths of 2959 bp, 2449 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.<br><br>The bands of pTerephthalate-B（~4000 bp、~2000 bp）from PCR are identical to the theoretical lengths of 4111 bp，2000 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.
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<figcaption>Fig.3  The bands of T7-tphB-tpaK（~2000+ bp）from PCR
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<br><br>The bands of T7-tphB-tpaK（~2000+ bp）from PCR are identical to the theoretical lengths of 2576 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.
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<figcaption>Fig.4  The bands of tphA2-tphA3-tphB -tphA1（~3000 bp）from PCR<br><br>The bands of tphA2-tphA3-tphB -tphA1（~3000 bp）from PCR are identical to the theoretical lengths of 2971 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.
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Latest revision as of 01:25, 2 October 2024


tphB

TphB is a dehydrogenase that oxidizes the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting in the production of PCA.

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 155
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 202
    Illegal NgoMIV site found at 877
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

TPA 1,2-dioxygenase (TPADO) is a two-component oxygenase consisting of three parts, TphA1, TphA2, and TphA3, which together enable TPADO to effectively catalyze the oxidative reaction of TPA, converting TPA to the intermediate product 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD). TphB is a dehydrogenase that oxidizes the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting 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 catalyzing the oxygenation reaction in the active site.TphA2 contains the active site in direct contact with the substrate, TPA, and contains the Cys-X1-His X17-Cys-X2-His pattern, binds to Rieske-type [2Fe-2S] iron-sulfur clusters and participates in electron transfer, which is a key part of the catalytic reaction of dioxygenases , TphA3 participates in the construction of the substrate channel or the appropriate positioning of the active site, and assists TphA2 in completing the oxidation reaction; TphA1 does not directly participate in the oxygenation reaction, but it 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 oxidized component of TPADO.

Fig 1. tph gene cluster core mechanism of converting TPA to PCA

Molecular cloning

Initially, we transformed the company-synthesized plasmids containing designed sequences into E. coli DH5α for amplification, allowing us to obtain a sufficient quantity of plasmid DNA for subsequent experiments. Following this, colony PCR was performed to confirm successful transformation, and the required plasmids were subsequently extracted for further experimentation.Subsequently, we employed PCR to obtain the target fragments, which were then integrated into the requisite plasmids for our study.

We constructed three plasmids for P. putida KT2440: pTerephthalate-A, pTerephthalate-B, and pRhamnolipid. We verified the size of each plasmid as well as all the fragments involved in constructing the plasmids .
Fig.2 The bands of pTerephthalate-A and pTerephthalate-B from PCR

The bands of pTerephthalate-A（~3000bp）from PCR are identical to the theoretical lengths of 2959 bp, 2449 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.

The bands of pTerephthalate-B（~4000 bp、~2000 bp）from PCR are identical to the theoretical lengths of 4111 bp，2000 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.
Fig.3 The bands of T7-tphB-tpaK（~2000+ bp）from PCR

The bands of T7-tphB-tpaK（~2000+ bp）from PCR are identical to the theoretical lengths of 2576 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.
Fig.4 The bands of tphA2-tphA3-tphB -tphA1（~3000 bp）from PCR

The bands of tphA2-tphA3-tphB -tphA1（~3000 bp）from PCR are identical to the theoretical lengths of 2971 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.