Difference between revisions of "Part:BBa K1850003"

Revision as of 22:01, 20 September 2015

pRha - fimH - SpyTag_225 - HisTag_258

Titratable Rhamnose promtoter with fimH gene and HisTag insertion at position 258 for nickel binding. SpyTag was added for use with binding partner Spycatcher but not experimentally validated. This part is for the production of modified type 1 pili.

Usage and Biology

This part contains the fimH adhesin under control of a titratable rhamnose promoter BBa_K902065. The FimH protein is a subunit of a naturally occurring structure in some strains of E. coli called type 1 pili. These hairlike appendages typically manifest as organelles on the surface of pathogenic E. coli which are responsible for urinary tract infections in humans. The FimH adhesin is found at the tip of the pilus, and binds naturally to the sugar mannose. This part can be cotransformed with BBa_K1850013, which contains the rest of the fim operon, and induced to produce type 1 pili.

Type 1 pili-producing strains of E. coli are able to clump together, or agglutinate, eukaryotic cells such as S. cervisiae (Baker's Yeast) by binding to the mannose sugar which is displayed on their cell surface. This behaviour is the basis of a standard assay used to detect pili production, see [http://2015.igem.org/Team:Harvard_BioDesign/Platform Agglutination Assay] for more information. BBa_K1850000, should be transferred to a low copy expression backbone, fimH Low Copy Expression Backbone and cotransformed with BBa_K1850013 according to its specifications into a strain that does not normally produce Type 1 Pili (see strain JW4275-1 from the [http://cgsc.biology.yale.edu/KeioList.php Keio Collection]). If it is then induced at an OD 600 of .3-.7 with .5% rhamnose and .01% arabinose overnight, it will recover the ability to agglutinate yeast that is missing from the chassis strain.

With the addition of a HisTag for measurability, induction of fimH with rhamnose under the pRha promoter showed increasing concentration of fimH on an anti-his Western Blot, see BBa_K1850006.

FimH can be modified to bind to a variety of biotic and abiotic surfaces by introducing a binding peptide with the desired affinity to site 225, 258 or the N terminal (recommended for folded binding motifs). These sites are highlighted in the following image orange, red and purple respectively:

Q5 PCR mutagenesis New England Biolabs was used for this purpose but other techniques may also be suitable. See our [http://2015.igem.org/Team:Harvard_BioDesign/Platform wiki] for more information. Chimeric fimH constructs were created that bind to Nickel, Stainless Steel, and Caco2 colorectal cancer cells have been experimentally validated. This part contains a 6xHisTag intended to bind to Nickel at the 258 insertion site and a SpyTag at the 225 location which is intended to form a covalent bond with SpyCatcher (Zakeri et. al 2012).

Sequence and Features

References

Zakeri, B., Fierer, J., Celik, E., Chittock, E., Schwarz-Linek, U., Moy, V., & Howarth, M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences.