Coding

Part:BBa_K4701202:Design

Designed by: Henri Sundquist   Group: iGEM23_Aalto-Helsinki   (2023-07-12)


FAST-PETase (Engineered PET hydrolase)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Codon optimization was applied with Geneious using E. coli K-12 codon usage tables. Restriction sites used in various BioBrick standards were removed with silent mutations. Lastly, some repeats were eliminated and GC-content decreased to allow lower complexity and synthesis by IDT. Note that the sequence doesn't include a start codon, as like in nature, our team prefixed the sequence with a signal peptide.


Source

The nucleotide sequence for IsPETase was retrieved from an I. sakaiensis sequencing contig (GenBank: BBYR01000074.1). The correct range was found by alignment against the protein sequence of IsPETase (UniProt: A0A0K8P6T7), excluding the first 27 residues which constitute a signal peptide. The five amino acid mutations were applied and the protein sequence was confirmed to align with the FAST-PETase sequence (PDB: 7SH6).

References