Difference between revisions of "Part:BBa K1850003"
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<partinfo>BBa_K1850003 short</partinfo> | <partinfo>BBa_K1850003 short</partinfo> | ||
| − | Titratable Rhamnose promtoter with fimH gene and HisTag insertion at position 258 for nickel binding. | + | 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. |
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===Usage and Biology=== | ===Usage and Biology=== | ||
| + | This part contains the ''fimH'' adhesin under control of a 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_K1850000, 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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| + | https://static.igem.org/mediawiki/parts/f/fb/Flocculation_with_inducers.png | ||
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| + | 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 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1850003 BBa_K1850003]. | ||
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| + | 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: | ||
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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. 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). | ||
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<partinfo>BBa_K1850003 parameters</partinfo> | <partinfo>BBa_K1850003 parameters</partinfo> | ||
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| + | ===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. | ||
Revision as of 19:27, 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.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.