Part:BBa_K5175031

T7 promoter-lac operator-pelB-MHETase-G4S-FAST-PETase-T7 terminator

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 826
  Illegal PstI site found at 1169
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2820
  Illegal PstI site found at 826
  Illegal PstI site found at 1169
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 709
  Illegal XhoI site found at 1967
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 826
  Illegal PstI site found at 1169
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 826
  Illegal PstI site found at 1169
  Illegal NgoMIV site found at 166
  Illegal NgoMIV site found at 508
  Illegal NgoMIV site found at 896
  Illegal NgoMIV site found at 1259
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

This composite component combines T7 promoter, lac operator and MHETase-PETase to enable the expression of MHETase-FAST-PETase specifically under IPTG induction, which is to introduced the MHETase-FAST-PETase dual enzyme system to enable engineered E.coli to degrade polyethylene terephthalate from polymers into monomers.

MHETase-FAST-PETase

PETase and MHETase are from the strain Ideonella sakaiensis 201-F6, and PET can be degraded by the synergistic action of the two enzymes. FAST-PETase is a machine-learning obtained PETase with properties suitable for in situ PET degradation at mild temperatures and moderate pH conditions . However, the main product of PETase degradation of PET is MHET, and the MHET intermediate tends to bind tightly to PET degrading enzyme in a non-catalytic pose, which leads to the inhibition of PET degrading enzyme. Therefore, an efficient MHET hydrolase is needed to degrade the intermediate product in time to further depolymerise MHET into its monomers terephthalic acid and ethylene glycol. Multi-enzyme systems promote substrate channeling and proximity effects between enzymes. This greatly reduces the diffusion limitation between enzyme active centres, thus promoting enzyme synergy and improving catalytic efficiency. In the process of constructing a dual enzyme system, we used bioinformatics to simulate the molecular docking of the linker connecting the two enzymes, and after simulation prediction, we chose the G4S flexible peptide as the linker of FAST-PETase and MHETase, and constructed the two into a dual enzyme system. We hoped that E.coli could exocytose the PETase-MHETase dual enzyme system to degrade PET microplastics in the environment. To this end, the pelB signal peptide was added to enhance the ability of BL21 to secrete MHETase-PETase.

Fig 1.Schematic diagram of FAST-PETase, MHETase dual enzyme system function

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 developed FAST-PETase-MHETase (pPM), MHETase-FAST-PETase (pMP) to independently validate their function.

Fig 2.The bands of pPM,pMP（2500+ bp）from PCR

The bands of pPM（2500+ bp）from PCR are identical to the theoretical lengths of 2682bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.