Part:BBa_K1850008:Design
pRha - fimH 136 KO - SpyTag_225 - HisTag_225
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 738
- 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 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.
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., Lars Pallesen, Hugh Connell, David L. Hasty, and Per Klemm. "Linker Insertion Analysis of the FimH Adhesin of Type 1 Fimbriae in an Escherichia Coli FimH-null Background." FEMS Microbiology Letters 137.2-3 (1996): 257-63. Oxford Journals. Federation of European Microbiological Societies, 1 Apr. 1996. Web. 18 Sept. 2015.