Difference between revisions of "Part:BBa K1850004"

Latest revision as of 20:39, 1 November 2017

pRha - fimH - SpyTag_N

Usage and Biology

This part contains the fimH adhesin with a HisTag fusion under control of the 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_K1850004 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.

FimH can be modified to bind to a variety of biotic and abiotic surfaces by introducing a binding peptide fusion 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. This part contains a SpyTag peptide at the N terminal which should bind to SpyCatcher but has not been characterized (Zakeri et. al 2012).

Exeter iGEM 2017 characterisation

As part of the Exeter iGEM 2107 project, Pili+, we wanted to characterise expression of the FimH protein modified at the N-terminal. We decided to change the SpyTag for a 6xHistidine tag to allow for easy characterisation of protein expression via SDS-PAGE and Western Blot. Please see our registry page which was created as we modified the DNA sequence for this part https://parts.igem.org/Part:BBa_K2324013.

Sequence and Features