Coding

Part:BBa_K1850011:Design

Designed by: Lydia Goldberg   Group: iGEM15_Harvard_BioDesign   (2015-09-15)

pRha - fimH 49 KO - RPMrel - SpyTag_225 - HisTag_225


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 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 changed site 49 on FimH, since researchers found that it knocked out mannose binding, but maintained expression of the FimH protein.

We selected a cancer-binding peptide called RPMrel from the TumorHoPe database. This peptide was used by the 2012 HKUST Hong Kong iGEM Team in their project on colon cancer. RPMrel was discovered using a technique called phage display and tested in vitro in a variety of cancer cell lines. It was tested with a payload of a mitochondrial toxin that selectively killed cancer cells.

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.

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.

Schembri, Mark A., Evgeni V. Sokurenko, and Per Klemm. "Functional Flexibility of the FimH Adhesin: Insights from a Random Mutant Library." Infection and Immunity 68.5 (2000): 2638-646. NCBI. American Society for Microbiology. Web. 18 Sept. 2015.

Kelly, Kimberly A., and David A. Jones. "Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection." Neoplasia 5.5 (2003): 437-44. NCBI. Neoplasia Press Inc. Web. 17 Sept. 2015.