Difference between revisions of "Part:BBa K3039006"
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===Usage and Biology===
 
===Usage and Biology===
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The enzymes PETase and MHETase were first discovered in Ideonella sakaiensis 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 (E. coli K12) codon optimized DNA of the R411A_S419G_F424N 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. These mutations have been reported in past papers to give MHETase the ability to degrade BHET.   
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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 <i>Escherichia coli</i> K12 (<i>E. coli</i> K12) codon optimized DNA of the R411A_S419G_F424N 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. These mutations have been reported in past papers to give MHETase the ability to degrade BHET.   
 
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The native predicted signal peptide (Met1-Ala19) was removed from the WT MHETase sequence (Palm et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The amino acid sequence was submitted to Twist Bioscience who codon optimised the sequence for E. coli, ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for potential TypeIIS assembly. The resulting CDS was synthesised and cloned, by Twist, into pET28. This added a 63 AA His-tag and thrombin cleavage site to the N-terminal of the protein, a T7 promoter and T7 terminator.  
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The native predicted signal peptide (Met1-Ala19) was removed from the WT MHETase sequence (Palm et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The amino acid sequence was submitted to Twist Bioscience who codon optimised the sequence for <i>E. coli</i>, ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for potential TypeIIS assembly. The resulting CDS was synthesised and cloned, by Twist, into pET28. This added a 63 AA His-tag and thrombin cleavage site to the N-terminal of the protein, a T7 promoter and T7 terminator.  
  
  

Revision as of 11:29, 2 September 2019

BHETase 2

Usage and Biology

The enzymes PETase and MHETase were first discovered in Ideonella sakaiensis 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 (E. coli K12) codon optimized DNA of the R411A_S419G_F424N 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. These mutations have been reported in past papers to give MHETase the ability to degrade BHET.

The native predicted signal peptide (Met1-Ala19) was removed from the WT MHETase sequence (Palm et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The amino acid sequence was submitted to Twist Bioscience who codon optimised the sequence for E. coli, ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for potential TypeIIS assembly. The resulting CDS was synthesised and cloned, by Twist, into pET28. This added a 63 AA His-tag and thrombin cleavage site to the N-terminal of the protein, a T7 promoter and T7 terminator.



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]