Part:BBa_K4701301:Design

lamB-MHETase

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 briefly

The wild-type protein sequence for MHETase was used. The native signal sequence was removed and replaced with the lamB signal sequence based on a recent study [1]. Codons were optimized with IDT, with BioBrick restriction sites being removed by additional tweaking. Later, repeats were removed and GC-content was reduced as initial synthesis failed.

Design in detail

The original nucleotide sequence of MHETase was acquired from the European Nucleotide Archive (ENA: GAP38911), and further confirmed by aligning against the reported protein sequence (UniProt: A0A0K8P8E7). The first 17 residues corresponding to the native signal were replaced with the lamB signal sequence followed by a methionine-alanine linker as done in the earlier study [1]. The nucleotide sequence for the signal was acquired by aligning sequence from the lambda receptor (ENA: M24997) against the protein sequence reported in the paper. MHETase sequence was codon optimized using IDTs tool with the E. coli K-12 codon usage tables. Again, some manual tweaking was required to remove BioBrick restriction sites, and to remove repeats to allow for synthesis. Next, a short Gly/Ser-linker was added downstream the MHETase sequence followed by a 6xHis-tag for purification. Lastly, a stop codon was added so that the part can be cloned independently into any expression vector. For our cloning strategy, the part was prefixed with “CAT” and suffixed with “CTCGAG” to add the NdeI and XhoI restriction sites respectively.

Before ordering synthesis of the part from IDT, a biophysical model which predicts translation rates based on the change in free energy of ribosome binding was used [2]. The results are shown in figure 1. Furthermore, the ordered insert was flanked on both sides by 30 base pairs of 50% GC-content stabilizing sequence to allow for restriction enzymes to act on their sites, and to allow the design of PCR primers for later.

Figure 2: Predicted translation on the relative arbitrary unit scale for BBa_K4701301 cloned into pRSFDuet-1 with NdeI and XhoI. Indexing starts from the first nucleotide after the upstream T7lac promoter. Translation rate for the two alternative start codons are roughly 6810 and 9910 respectively.

For a more detailed description of the iterative design process, refer to our wiki.

References

[1] Sagong, H., et al (2022). Decomposition of the PET Film by MHETase Using Exo-PETase Function. ACS Catalysis. 10(8), 4805–4812. https://doi.org/10.1021/acscatal.9b05604

[2] Salis, HM., et al. (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nature Biotechnology. 27(10), 946–950. https://doi.org/10.1038/nbt.1568