Difference between revisions of "Part:BBa K5175030"

 
 
(3 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K5175030 short</partinfo>
 
<partinfo>BBa_K5175030 short</partinfo>
  
−
It is a composite component consisting of the T7 promoter, T7 terminator, target genes tphA2, tphA3, tphA1, tphB. It is responsible for converting TPA to 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD) and oxidising the diol moiety (two hydroxyl groups) of DCD to a keto group to result in the production of PCA .
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 17: Line 16:
 
<partinfo>BBa_K5175030 parameters</partinfo>
 
<partinfo>BBa_K5175030 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
<h1>Description</h1>
 +
This composite component combines T7 promoter, lac operator and PETase-MHETase to enable the expression of FAST-PETase-MHETase specifically under IPTG induction, which is to introduced the FAST-PETase-MHETase dual enzyme system to enable engineered <i>E.coli</i> to degrade polyethylene terephthalate from polymers into monomers.
 +
<h1>FAST-PETase-MHETase</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 .
 +
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.
 +
<html>
 +
<figure><center>
 +
<img
 +
alt=""
 +
src="https://static.igem.wiki/teams/5175/resources/design-2/design-01.png"
 +
width="700"
 +
title="">
 +
<figcaption>Fig 1.Schematic diagram of FAST-PETase, MHETase dual enzyme system function</figcaption>
 +
</figure>
 +
<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.
 +
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.
 +
<html>
 +
<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 06:44, 2 October 2024


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


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1645
    Illegal PstI site found at 1988
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2820
    Illegal PstI site found at 1645
    Illegal PstI site found at 1988
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1528
    Illegal XhoI site found at 2786
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1645
    Illegal PstI site found at 1988
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1645
    Illegal PstI site found at 1988
    Illegal NgoMIV site found at 166
    Illegal NgoMIV site found at 1327
    Illegal NgoMIV site found at 1715
    Illegal NgoMIV site found at 2078
  • 1000
    COMPATIBLE WITH RFC[1000]


Description

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

FAST-PETase-MHETase

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.

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.