Difference between revisions of "Part:BBa K1850002"
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<partinfo>BBa_K1850002 short</partinfo> | <partinfo>BBa_K1850002 short</partinfo> | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
| + | This part contains the ''fimH'' adhesin with a HisTag fusion under control of the titratable rhamnose promoter [https://parts.igem.org/Part:BBa_K902065 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 <partinfo>BBa_K1850013</partinfo>, which contains the rest of the ''fim'' operon, and induced to produce type 1 pili. | ||
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| + | 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_K1850002 should be transferred to a low copy expression backbone, [https://benchling.com/s/A9YgHe9r/edit fimH Low Copy Expression Backbone] and cotransformed with <partinfo>BBa_K1850013</partinfo> 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. | ||
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| + | <img src="https://static.igem.org/mediawiki/parts/f/fb/Flocculation_with_inducers.png" height="475px" width="750px"/> | ||
| + | </body></html> | ||
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| + | 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: | ||
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| + | https://static.igem.org/mediawiki/2015/2/22/Harvard_Fim_Animated.gif | ||
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| + | Q5 PCR mutagenesis [https://www.neb.com/products/e0554-q5-site-directed-mutagenesis-kit 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 site 225 which should bind to SpyCatcher but has not been characterized (Zakeri et. al 2012). | ||
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Latest revision as of 17:42, 21 September 2015
pRha - fimH - SpyTag_225
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_K1850002 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 site 225 which should bind to SpyCatcher but has not been characterized (Zakeri et. al 2012).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]