Difference between revisions of "Part:BBa K3039007"

Revision as of 23:00, 21 October 2019

SP_lamB-PETase

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 wild type PETase with a lamB signal peptide and N-terminal His-tag. 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 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.

References

{1] Hyeoncheol Francis Son, In Jin Cho, Seongjoon Joo, Hogyun Seo, Hye-Young Sagong, So Young Choi, Sang Yup Lee, Kyung-Jin Kim; Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation (2019) ACS Catal. 9(4), 3519-3526

[2] Congcong Liua, Chao Shia, Sujie Zhua, Risheng Weia, Chang-Cheng Yin; Structural and functional characterization of polyethylene terephthalate hydrolase from Ideonella sakaiensis (2019) Biochem. Biophys. Res. Commun. 508(1), 289-294

[3] 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 654
1000
COMPATIBLE WITH RFC[1000]

@@ Line 8: / Line 8: @@
 <br />
 <br />
-This sequence is the <i>Escherichia coli</i> K12 (<i>E. coli</i> K12) codon optimized DNA of wild type PETase with a lamB signal peptide and N-terminal His-tag 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.
+This sequence is the <i>Escherichia coli</i> K12 (<i>E. coli</i> K12) codon optimized DNA of wild type PETase with a lamB signal peptide and N-terminal His-tag. 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.
 <br />
 <br />
-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, pX1800 (University of Exeter).
 ===Characterisation===