Difference between revisions of "Part:BBa K5175030"
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<h1>Description</h1> | <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 E.coli to degrade polyethylene terephthalate from polymers into monomers. | + | 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> | <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 . | 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. | ||
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| − | + | <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> | <h1>Molecular cloning</h1> | ||
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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. | ||
Latest revision as of 06:44, 2 October 2024
T7 promoter-lac operator-pelB-FAST-PETase-G4S-MHETase-T7 terminator
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1645
Illegal PstI site found at 1988 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2820
Illegal PstI site found at 1645
Illegal PstI site found at 1988 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1528
Illegal XhoI site found at 2786 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1645
Illegal PstI site found at 1988 - 25INCOMPATIBLE 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 - 1000COMPATIBLE 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.
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.
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.