Difference between revisions of "Part:BBa K3039016"
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===Usage and Biology===
 
===Usage and Biology===
 
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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 S416A_F424N
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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.  
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mutant MHETase with the ompA signal peptide and a His tag attached. The ompA signal peptide has been used to secrete the enzyme into the periplasmic domain of <I>E.coli</I> and the His tag was attached in order to more easily identify the enzymes. The wild type MHETase doesn’t show BHET degrading activity. These mutation has been reported in past papers to give MHETase the ability to degrade BHET.
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This sequence is the <i>Escherichia coli</i> K12 (<i>E. coli</i> K12) codon optimized DNA of the S416A_F424N mutant MHETase with the ompA signal peptide and N-terminal His-tag. The ompA signal peptide has been used to secrete the enzyme into the periplasmic domain of <i>E.coli</i> when modified <i>E.coli</i> is added to the filter system and 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 the literature to give MHETase the ability to degrade BHET (Gottfried et al 2019).  
 
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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 21 AA ompA signal peptide, which allows for localisation of the enzyme within the periplasm (Fischer et al 1993) 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).
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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 21 AA ompA signal peptide, which allows for localisation of the enzyme within the periplasm (Fischer et al 1993) 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, pX1800 (University of Exeter).
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===Characterisation===
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In order to characterise our part and determine the rate of its activity and prove its functionality we intended to run a series of experiments. However, due to the change in design of our experiments, excreted protein was no longer required, and experimentation with this part was not completed.
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===References===
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[1] Gottfried J. Palm, Lukas Reisky, Dominique Böttcher, Henrik Müller, Emil A. P. Michels, Miriam C. Walczak, Leona Berndt, Manfred S. Weiss, Uwe T. Bornscheuer & Gert Weber; Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate (2019) Nat. Commun. 10(1717)
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[2] Bernhard Fischer, Barry Perry, Gareth Phillips, lan Sumner, Peter Goodenough; Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli (1993) Appl. Microbiol. Biotechnol. 39(570) 
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<span class='h3bb'>Sequence and Features</span>-->
  
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===Sequences and Features===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3039016 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3039016 SequenceAndFeatures</partinfo>
  

Latest revision as of 23:46, 21 October 2019


SP_ompA-MHETase S416A_F424N

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.

This sequence is the Escherichia coli K12 (E. coli K12) codon optimized DNA of the S416A_F424N mutant MHETase with the ompA signal peptide and N-terminal His-tag. The ompA signal peptide has been used to secrete the enzyme into the periplasmic domain of E.coli when modified E.coli is added to the filter system and 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 the literature to give MHETase the ability to degrade BHET (Gottfried et al 2019).

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 21 AA ompA signal peptide, which allows for localisation of the enzyme within the periplasm (Fischer et al 1993) 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, pX1800 (University of Exeter).

Characterisation

In order to characterise our part and determine the rate of its activity and prove its functionality we intended to run a series of experiments. However, due to the change in design of our experiments, excreted protein was no longer required, and experimentation with this part was not completed.



References

[1] Gottfried J. Palm, Lukas Reisky, Dominique Böttcher, Henrik Müller, Emil A. P. Michels, Miriam C. Walczak, Leona Berndt, Manfred S. Weiss, Uwe T. Bornscheuer & Gert Weber; Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate (2019) Nat. Commun. 10(1717)

[2] Bernhard Fischer, Barry Perry, Gareth Phillips, lan Sumner, Peter Goodenough; Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli (1993) Appl. Microbiol. Biotechnol. 39(570)


Sequences and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 600
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]