Coding

Part:BBa_K2695002:Design

Designed by: Sophie Hodson   Group: iGEM18_Exeter   (2018-09-18)


N. defluvii chlorite dismutase


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 site found at 8
    Illegal BsaI.rc site found at 861


Design Notes

The sequence was obtained from Nitrospira defluvii (NCBI accession number: ACE75544.1) and codon optimised for E. coli. It contains an N-terminal signal peptide which directs the protein to the periplasm. A His-tag was included, with flexible linkers either side, to allow for Western Blot analysis. The flexible linkers were included to prevent occlusion of the His-tag by the protein as it folds.

To construct the chlorite dismutase gene, the following linear DNA parts were assembled using ‘one pot’ modular cloning:

  • An inducible T7 promoter. This was chosen because it a well characterised strong promoter which is inducible with IPTG. This was important as we wanted to induce expression when the cells had reached mid-log phase, so that they would have grown enough in their new environment to produce a lot of protein. This part has an inbuilt ribosome binding site, so we did not need to add one.
  • The N. defluvii chlorite dismutase coding sequence.
  • The B0015 terminator. This was chosen because it is the most commonly used terminator in iGEM. It is RFC10 compatible and can be found on the iGEM parts registry (https://parts.igem.org/Part:BBa_B0015)

Source

This part was synthesised by IDT using the sequence from NCBI. NCBI reference number for N. defluvii chlorite dismutase: ACE75544.1