Coding

Part:BBa_K1850003:Design

Designed by: Lydia Goldberg   Group: iGEM15_Harvard_BioDesign   (2015-09-15)
Revision as of 21:38, 18 September 2015 by Lgoldberg (Talk | contribs) (→‎Source)


pRha - fimH - SpyTag_225 - HisTag_258


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

We selected a rhamnose-inducible promoter (BBa_K902065) with a strong ribosome binding site (BBa_B0034), since this promoter is titratable and would allow for controlled expression of the fimH adhesin.

We edited out an illegal PstI cut site in fimH through site-directed mutagenesis.

The SpyTag binding motif was inserted into the fusion site of fimH via site-directed mutagenesis.

The nickel binding HisTag was inserted into fusion sites of fimH via site-directed mutagenesis.

We picked sites 225 and 258 since there was strong evidence in the literature that small fusions inserted at these sites such as his-tags were expressed and functional on assembled pili.

Source

The fimH gene was amplified from the E. coli K-12 genome.

References