Part:BBa_K1850003:Design
pRha - fimH - SpyTag_225 - HisTag_258
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
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.