Part:BBa_K5175039

T7 promoter-tpaK-T7 terminator

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 919
  Illegal PstI site found at 1208
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1475
  Illegal PstI site found at 919
  Illegal PstI site found at 1208
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 919
  Illegal PstI site found at 1208
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 919
  Illegal PstI site found at 1208
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

It is a composite component consisting of the T7 promoter, T7 terminator, target genes tpaK. It is responsible for transporting TPA.

Usage and Biology

In Comamonas sp. E6, the TPA transport system resembles a three-part tripartite tricarboxylate transporter (TTT), which consists of three components, TphC, TpiA, and TpiB, with TphC acting as a Substrate-Binding TphC specifically recognizes and binds TPA as a Substrate-Binding Protein (SBP); TpiA and TpiB are transmembrane proteins that form part of the transporter protein complex, whereas TphC delivers TPA from the periplasm (extracellular space) to these membrane proteins. Thus only when tphC and tpiA and tpiB genes are introduced simultaneously, our engineered bacteria can transport TPA . While TpaK, another TPA transporter protein encoded in Rhodococcus sp. RHA1, does not require the presence of tpiA or tpiB to function as a transporter, we chose TpaK as the TPA transporter protein to be applied in engineering P.putida.

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 P. putida through electroporation. Given that our wild-type P. 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 tpaK（~1500 bp）from PCR

The bands of tpaK（~1500 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.