Part:BBa_K5117010

BhrPET

gene of the uncultured bacterium HR29 encoding a polyethylene terephthalate hydrolase (PETase, EC 3.1.1.101), a homologue of the leaf-branch compost cutinase (LCC), codon optimized for Bacillus subtilis (Xi et al. 2021), but additionally including the signal peptide of the aprE gene for secretion in Bacillus subtilis

BhrPET only served for design purposes of the TU Dresden iGEM 2024 Team and was required for the construction of composite parts (see Contribution page).

Target organism: Bacillus subtilis

Main purpose of use: Gene expression and protein production using the host Bacillus subtilis

Design

For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, XbaI, SpeI, PstI and NotI were removed from the coding sequence. To make the part compatible with the Type IIS standard, BsaI and SapI sites were removed as well. This was achieved by codon exchange using the codon usage table of Bacillus subtilis (Codon Usage Database Kazusa).

Sequence 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 579
  Illegal AgeI site found at 624
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

Xi X., Ni K., Hao H., Shang Y., Zhao B., Qian Z. (2021): Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29. Enzyme and microbial technology 143, 109715.