Coding

Part:BBa_K1850007:Design

Designed by: Lydia Goldberg   Group: iGEM15_Harvard_BioDesign   (2015-09-15)
Revision as of 21:51, 18 September 2015 by Lgoldberg (Talk | contribs)

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pRha - fimH - MBD_N - SpyTag_225


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 374
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 374
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 374
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 374
  • 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 stainless steel binding Metal Binding Domain (MBD) was inserted into fusion sites of fimH via site-directed mutagenesis.

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

We picked the N-terminus as a new insertion site to try since prior work did not show if peptide fusions could be expressed and properly assembled at this site.

Source

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

References

Zakeri, Bijan, Jacob O. Fierer, Emrah Celik, Emily C. Chittock, Ulrich Schwarz-Linek, Vincent T. Moy, and Mark Howarth. "Peptide Tag Forming a Rapid Covalent Bond to a Protein, through Engineering a Bacterial Adhesin." Proceedings of the National Academy of the United States of America 109.12 (2012): E690-697. PNAS. National Academy of the Sciences. Web. 18 Sept. 2015.

Nguyen, P. Q. et al. Programmable biofilm-based materials from engineered curli nanofibres. Nat. Commun. 5:4945 doi: 10.1038/ncomms5945 (2014).

Pallesen, Lars, Lars K. Poulsen, Gunna Christiansen, and Per Klemm. "Chimeric FimH Adhesin of Type 1 Fimbriae: A Bacterial Surface Display System for Heterologous Sequences." Microbiology 141 (1995): 2839-848. SGM Journals. Society for General Microbiology, 01 Nov. 1995. Web. 18 Sept. 2015.

Bhomkar, Prasanna, Wayne Materi, Valentyna Semenchenko, and David S. Wishart. "Transcriptional Response Of E. Coli Upon FimH-mediated Fimbrial Adhesion." Gene Regulation and Systems Biology 4 (2010): 1-17. NCBI. Libertas Academica, 24 Mar. 2010. Web. 18 Sept. 2015.