Coding

Part:BBa_K2695004:Design

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


A. suillum signal peptide for perchlorate reductase (subunit C)


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 106
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 8
    Illegal BsaI.rc site found at 810


Design Notes

We used signal peptides from subunits A and C only as the other 2 subunits (B and D) do not have signal peptides.
We inserted the signal peptide from: A. suillum PcrA. This was synthesised by IDT along with the T7 promoter from plasmid pET21 (Novagen) and the B0015 terminator, and a modular cloning strategy (http://2018.igem.org/Team:Exeter/Protocols) was used to build the complete genes in pSB1C3. As the T7 promoter contains an XbaI site the complete genes have not been submitted to the iGEM registry. Instead the coding sequences, in pSB1C3, have been submitted (http://2018.igem.org/Team:Exeter/Parts) for future teams to use with their own chosen promoters.

The sequence was obtained from Azospira suillum PcrC (NCBI accession number for GFP: PBD-2B3P) 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 signal peptide GFP 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 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 found on the NCBI database. NCBI reference number for A. suillum genome: CP003153

References