Part:BBa_K5175035

T7 promoter- tphA2-tphA3 -tphA1-T7 terminator

Sequence and Features

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 1951
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2826
  Illegal NheI site found at 2891
  Illegal PstI site found at 949
  Illegal PstI site found at 982
  Illegal PstI site found at 1951
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 426
  
• 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 1951
  
• 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 1951
  Illegal NgoMIV site found at 477
  Illegal NgoMIV site found at 1516
  Illegal NgoMIV site found at 2266
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

It is a composite component consisting of the T7 promoter, T7 terminator, target genes tphA2, tphA3, tphA1. It is responsible for converting TPA to 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD) adding a new exogenous pathway for TPA absorbtion. Thus, TPA could serve as a new carbon source to provide energy for P.putida.

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

TphA2 constitute the large subunits of the TPADO oxidase component responsible for binding to the TPA substrate and catalyzing the oxygenation reaction in the active site.

TphA3

TphA3 constitutes the small subunits of the TPADO oxidase component responsible for binding to the TPA substrate and catalyzing the oxygenation reaction in the active site.

TphA1

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 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 . 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-tphA2-tphA3 -tphA1-T7（~3000 bp）from PCR

The bands of T7-tphA2-tphA3 -tphA1-T7（~3000 bp）from PCR are identical to the theoretical lengths of 2959 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.