Part:BBa_K4701210:Design

Engineered L-1,2-propanediol oxidoreductase (fucO)

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 25
  

Design Notes

fucO contains a double-methionine start meaning that there are two alternative ORFs. We chose to omit the first methionine as in UniProt. This means that the indexing of the swapped residues were shifted from I7L/L8V [1] to I6L/L7V and applied. As the genes originate from E. coli which is also the target host, only modest codon optimization was applied with IDTs tool using usage tables for E. coli K-12. Sites in in BioBrick standards were eliminated as much as possible.

As this CDS would be expressed together with aldA in a pRSFDuet-1 dual expression vector, we needed to balance predicted translation rates of the two ORFs from the two multiple cloning sites. This was done by manually tweaking 5' end of the sequence to introduce secondary structure reducing the change in free energy during ribosome binding, which is a predictor of translation rate [2].

For more information on the tandem design process with BBa_K4701211 visit our wiki.

Source

The nucleotide sequence of fucO was acquired from GenBank: M31059 by aligning against the protein sequence (UniProt: P0A9S1).

References

[1] Pandit, AV., et al (2021) Engineering Escherichia coli for the utilization of ethylene glycol. Microbial Cell Factories. 20(1), 22. https://doi.org/10.1186/s12934-021-01509-2

[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