Difference between revisions of "Part:BBa K3039008"
 
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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 <i>Escherichia coli</i> K12 (<i>E. coli</i> K12) codon optimized DNA of the R208A mutant of PETase with the lamB signal peptide and His tag attached. The His tag was attached in order to more easily identify the enzymes. This mutation has been reported in past papers to increase the activity of PETase. The lamB signal peptide has been used in order to secrete the enzyme outside <i>E.coli</i>.  
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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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This sequence is the <i>Escherichia coli</i> K12 (<i>E. coli</i> K12) codon optimized DNA of the R208A mutant of PETase with the lamB signal peptide and His tag attached. The His tag was attached in order to more easily identify the enzymes. This mutation has been reported in the literature to increase the activity of PETase (Seo et al 2019). The lamB signal peptide has been used for extracellular production of the enzyme when modified <i>E.coli</i> is added to the filter system and the His-tag is attached to more easily identify the enzymes.  
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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).
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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, pX1800 (University of Exeter).
  
 
===Characterisation===
 
===Characterisation===
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In order to characterise our part and determine the rate of its activity and prove its functionality we have run a series of experiments. After transforming the Arctic Express, Rosetta Gami and BL21 DE3 strains of E. coli with our plasmid we induced the expression of the enzymes using IPTG. In order to confirm that the enzyme expression has been successful we ran a western blot which showed the presence of the enzyme in the soluble fractions of the sonicated cells. Afterwards the enzyme was purified and used in assays to show its functionality and determine the rate of its activity.
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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===
 
===References===
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[1]Seongjoon Joo, In Jin Cho, Hogyun Seo, Hyeoncheol Francis Son, Hye-Young Sagong, Tae Joo Shin, So Young Choi, Sang Yup Lee & Kyung-Jin Kim; Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation (2018) Nat. Commun. 9(382)  
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[1] Seongjoon Joo, In Jin Cho, Hogyun Seo, Hyeoncheol Francis Son, Hye-Young Sagong, Tae Joo Shin, So Young Choi, Sang Yup Lee & Kyung-Jin Kim; Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation (2018) Nat. Commun. 9(382)  
 
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[2]Hogyun Seo, Seongmin Kim, Hyeoncheol Francis Son, Hye-Young Sagong, Seongjoon Joo, Kyung-Jin Kim; Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli (2019) Biochem. Biophys. Res. Commun. 508(1), 250-255   
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[2] Hogyun Seo, Seongmin Kim, Hyeoncheol Francis Son, Hye-Young Sagong, Seongjoon Joo, Kyung-Jin Kim; Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli (2019) Biochem. Biophys. Res. Commun. 508(1), 250-255   
  
  

Latest revision as of 23:43, 21 October 2019

SP_lamB-PETase R280A

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 R208A mutant of PETase with the lamB signal peptide and His tag attached. The His tag was attached in order to more easily identify the enzymes. This mutation has been reported in the literature to increase the activity of PETase (Seo et al 2019). The lamB signal peptide has been used for extracellular production of the enzyme when modified E.coli is added to the filter system and the His-tag is attached to more easily identify the enzymes.

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, 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] Seongjoon Joo, In Jin Cho, Hogyun Seo, Hyeoncheol Francis Son, Hye-Young Sagong, Tae Joo Shin, So Young Choi, Sang Yup Lee & Kyung-Jin Kim; Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation (2018) Nat. Commun. 9(382)

[2] Hogyun Seo, Seongmin Kim, Hyeoncheol Francis Son, Hye-Young Sagong, Seongjoon Joo, Kyung-Jin Kim; Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli (2019) Biochem. Biophys. Res. Commun. 508(1), 250-255


Sequences and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 540
  • 1000
    COMPATIBLE WITH RFC[1000]