Difference between revisions of "Part:BBa K5175002"
 
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<h1>'''Usage and Biology'''</h1>
 
<h1>'''Usage and Biology'''</h1>
−
    PETase and MHETase are from the strain <i>Ideonella sakaiensis</i> 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 .
+
PETase and MHETase are from the strain <i>Ideonella sakaiensis</i> 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.
+
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 <i>E.coli</i> 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 PETase-MHETase.
 
We hoped that <i>E.coli</i> 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 PETase-MHETase.
  
 
<h1>'''Molecular cloning'''</h1>
 
<h1>'''Molecular cloning'''</h1>
−
    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.
+
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.
 
Subsequently, we employed PCR to obtain the target fragments, which were then integrated into the requisite plasmids for our study.
 
We constructed two plasmids, pPeteg-P and pPeteg-M, for <i>E. coli</i> BL21 to assess the activity of different dual enzyme systems in engineered <i>E. coli</i>. We verified the size of the plasmids as well as all the fragments involved in constructing the plasmids . In addition to pPeteg-P and pPeteg-M, we also developed FAST-PETase-MHETase (pPM), MHETase-FAST-PETase (pMP), and T7-fucO-aldA (pEG) to independently validate their function.
 
We constructed two plasmids, pPeteg-P and pPeteg-M, for <i>E. coli</i> BL21 to assess the activity of different dual enzyme systems in engineered <i>E. coli</i>. We verified the size of the plasmids as well as all the fragments involved in constructing the plasmids . In addition to pPeteg-P and pPeteg-M, we also developed FAST-PETase-MHETase (pPM), MHETase-FAST-PETase (pMP), and T7-fucO-aldA (pEG) to independently validate their function.
 +
<html>
 +
 +
<figure><center>
 +
<img
 +
alt=""
 +
src="https://static.igem.wiki/teams/5175/resources/result/result-01.png"
 +
width="700"
 +
title="">
 +
<figcaption>Fig 1.The bands of pPeteg-P (upper band) and pPeteg-M (lower band)（~3000 bp）from PCR<br>
 +
<br>The bands of pPeteg-P (upper band) and pPeteg-M (lower band)（~3000 bp）from PCR are identical to the theoretical lengths of 2862 bp estimated by the designed primer locations (homologous recombination fragments), which could demonstrate that these plasmids had successfully been obtained.</figcaption>
 +
</figure>
 +
<figure><center>
 +
<img
 +
alt=""
 +
src="https://static.igem.wiki/teams/5175/resources/result/result-02.png"
 +
width="700"
 +
title="">
 +
<figcaption>Fig 2.The bands of pPM,pMP（2500+ bp）from PCR<br>
 +
<br>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.</figcaption>
 +
</figure>

Latest revision as of 22:36, 1 October 2024


PETase-G4S-MHETase


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1536
    Illegal PstI site found at 1879
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1536
    Illegal PstI site found at 1879
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1419
    Illegal XhoI site found at 2677
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1536
    Illegal PstI site found at 1879
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1536
    Illegal PstI site found at 1879
    Illegal NgoMIV site found at 57
    Illegal NgoMIV site found at 1218
    Illegal NgoMIV site found at 1606
    Illegal NgoMIV site found at 1969
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

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 PETase-MHETase.

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 two plasmids, pPeteg-P and pPeteg-M, for E. coli BL21 to assess the activity of different dual enzyme systems in engineered E. coli. We verified the size of the plasmids as well as all the fragments involved in constructing the plasmids . In addition to pPeteg-P and pPeteg-M, we also developed FAST-PETase-MHETase (pPM), MHETase-FAST-PETase (pMP), and T7-fucO-aldA (pEG) to independently validate their function.

Fig 1.The bands of pPeteg-P (upper band) and pPeteg-M (lower band)（~3000 bp）from PCR

The bands of pPeteg-P (upper band) and pPeteg-M (lower band)（~3000 bp）from PCR are identical to the theoretical lengths of 2862 bp estimated by the designed primer locations (homologous recombination fragments), which could demonstrate that these plasmids had successfully been obtained.
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.