Difference between revisions of "Part:BBa K3039006"
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The enzymes PETase and MHETase were first discovered in <I>Ideonella sakaiensis</I>in 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles. The enzymes would be secreted into a filter that captures the microfibres. This sequence is the Escherichia coli K12 (<I>E. coli</I> K12) codon optimized DNA of the W397A mutant MHETase, with an attached His tag. The His tag was attached in order to more easily identify the enzymes. The wild type MHETase doesn’t show BHET degrading activity. This mutation has been reported in past papers to give MHETase the ability to degrade BHET.   
 
The enzymes PETase and MHETase were first discovered in <I>Ideonella sakaiensis</I>in 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles. The enzymes would be secreted into a filter that captures the microfibres. This sequence is the Escherichia coli K12 (<I>E. coli</I> K12) codon optimized DNA of the W397A mutant MHETase, with an attached His tag. The His tag was attached in order to more easily identify the enzymes. The wild type MHETase doesn’t show BHET degrading activity. This mutation has been reported in past papers to give MHETase the ability to degrade BHET.   
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The native predicted signal peptide (Met1-Ala33) was removed from the WT PETase sequence (Seo et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The 25 AA lamB signal peptide, which allows for excretion of the enzyme via the Sec-dependent translocation pathway (Seo et al 2019)* was added to the N-terminal followed by a 13 AA His-tag. The entire amino acid sequence was codon optimised for <I>E. coli</I> by IDT’s on-line Codon Optimisation tool ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for TypeIIS assembly. The iGEM TypeIIS prefix and suffix were added and DNA was synthesised by IDT as a double stranded g-block. TypeIIS assembly was used to clone the resulting CDS with the T7-promoter and B0015 terminator into a high-copy number, ampicillin vector, pX1900 (University of Exeter).
 
The native predicted signal peptide (Met1-Ala33) was removed from the WT PETase sequence (Seo et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The 25 AA lamB signal peptide, which allows for excretion of the enzyme via the Sec-dependent translocation pathway (Seo et al 2019)* was added to the N-terminal followed by a 13 AA His-tag. The entire amino acid sequence was codon optimised for <I>E. coli</I> by IDT’s on-line Codon Optimisation tool ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for TypeIIS assembly. The iGEM TypeIIS prefix and suffix were added and DNA was synthesised by IDT as a double stranded g-block. TypeIIS assembly was used to clone the resulting CDS with the T7-promoter and B0015 terminator into a high-copy number, ampicillin vector, pX1900 (University of Exeter).
  

Revision as of 09:33, 30 August 2019

BHETase 2

Usage and Biology

The enzymes PETase and MHETase were first discovered in Ideonella sakaiensisin 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles. The enzymes would be secreted into a filter that captures the microfibres. This sequence is the Escherichia coli K12 (E. coli K12) codon optimized DNA of the W397A mutant MHETase, with an attached His tag. The His tag was attached in order to more easily identify the enzymes. The wild type MHETase doesn’t show BHET degrading activity. This mutation has been reported in past papers to give MHETase the ability to degrade BHET.


The native predicted signal peptide (Met1-Ala33) was removed from the WT PETase sequence (Seo et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The 25 AA lamB signal peptide, which allows for excretion of the enzyme via the Sec-dependent translocation pathway (Seo et al 2019)* was added to the N-terminal followed by a 13 AA His-tag. The entire amino acid sequence was codon optimised for E. coli by IDT’s on-line Codon Optimisation tool ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for TypeIIS assembly. The iGEM TypeIIS prefix and suffix were added and DNA was synthesised by IDT as a double stranded g-block. TypeIIS assembly was used to clone the resulting CDS with the T7-promoter and B0015 terminator into a high-copy number, ampicillin vector, pX1900 (University of Exeter).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1104
    Illegal PstI site found at 653
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1104
    Illegal PstI site found at 653
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1104
    Illegal BamHI site found at 561
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1104
    Illegal PstI site found at 653
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1104
    Illegal PstI site found at 653
    Illegal NgoMIV site found at 246
    Illegal AgeI site found at 1130
  • 1000
    COMPATIBLE WITH RFC[1000]