Difference between revisions of "Part:BBa K3039014"
CeilidhWelsh (Talk | contribs) |
CeilidhWelsh (Talk | contribs) |
||
Line 4: | Line 4: | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
<br> | <br> | ||
− | 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 wild type 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 E.coli and the His tag was attached in order to more easily identify the enzymes. | + | 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 wild type 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. |
<br> | <br> | ||
<br> | <br> | ||
− | 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, pX1900 (University of Exeter). | + | 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). |
Revision as of 11:22, 30 August 2019
SP_ompA-MHETase
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 wild type 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 E.coli and the His tag was attached in order to more easily identify the enzymes.
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, pX1900 (University of Exeter).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 600
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]