Part:BBa_K5175036

T7 promoter - tphB-tpaK-T7 terminator

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1906
  Illegal PstI site found at 2195
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2462
  Illegal PstI site found at 1906
  Illegal PstI site found at 2195
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 242
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1906
  Illegal PstI site found at 2195
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1906
  Illegal PstI site found at 2195
  Illegal NgoMIV site found at 289
  Illegal NgoMIV site found at 964
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

It is a composite component consisting of the T7 promoter, T7 terminator, target genes tphB, tpaK. It is responsible for oxidising the diol moiety (two hydroxyl groups) of DCD to a keto group to result in the production of PCA and transporting TPA.

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). 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.

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.

TpaK

tpaK is a TPA transporter protein does not require other proteins for TPA transportation

Fig 1. TPA transport pattern of TphC and TpaK

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 . The plasmids were successfully introduced into *Pseudomonas putida* through electroporation. Given that our wild-type Pseudomonas putida exhibits resistance to chloramphenicol, the plasmids incorporated a kanamycin resistance marker. Consequently, we employed dual antibiotic selection plates to effectively screen for successfully transformed engineered strains.

Fig.2 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.