Part:BBa_K5175033
T7 promoter-lac operator-pelB-FAST-PETase-G4S-MHETase-Terminator-T7 promoter-fucO-aldA-T7 termina7 t
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1645
Illegal PstI site found at 1988
Illegal PstI site found at 3117
Illegal PstI site found at 4865
Illegal PstI site found at 5578 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2820
Illegal NheI site found at 2876
Illegal NheI site found at 5636
Illegal PstI site found at 1645
Illegal PstI site found at 1988
Illegal PstI site found at 3117
Illegal PstI site found at 4865
Illegal PstI site found at 5578 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1528
Illegal BamHI site found at 4403
Illegal XhoI site found at 2786 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1645
Illegal PstI site found at 1988
Illegal PstI site found at 3117
Illegal PstI site found at 4865
Illegal PstI site found at 5578 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1645
Illegal PstI site found at 1988
Illegal PstI site found at 3117
Illegal PstI site found at 4865
Illegal PstI site found at 5578
Illegal NgoMIV site found at 166
Illegal NgoMIV site found at 1327
Illegal NgoMIV site found at 1715
Illegal NgoMIV site found at 2078
Illegal AgeI site found at 3833
Illegal AgeI site found at 4034
Illegal AgeI site found at 4675 - 1000COMPATIBLE WITH RFC[1000]
Description
It is a composite component consisting of the T7 promoter, lac operator, target genes PETase-MHETase, fucO, aldA. It is responsible for enabling E.coli to degrade PET and increasing E.coli 's ability to efficiently utilise EG. FAST-PETase-MHETase dual enzyme system is introduced to enable engineered E.coli to degrade polyethylene terephthalate from polymers into monomers. While EG is first converted in E.coli to glycolaldehyde (GLA) by L-1,2 -propylene glycol oxidoreductase, which is subsequently converted to glycolic acid (GA) by aldehyde dehydrogenase A. GA can be metabolized by condensation with acetyl coenzyme A via the glyoxalate shunt to form malic acid. GA can also enter the metabolic pathway of H. coli by condensing with succinate via isocitrate lyase (encoded by the aceA gene), forming isocitrate
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.
fucO
fucO is the gene for L-1,2-propanediol oxidoreductase, which is an iron-dependent group III dehydrogenase and can convert Ethylene glycol(EG) to glycolaldehyde (GLA).
aldA
aldA is the gene for aldehyde dehydrogenase A, which is an enzyme with a relatively broad substrate specificity for small hydroxyaldehyde substrates and can convert glycolaldehyde (GLA) to glycolic acid (GA).
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.
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