Difference between revisions of "Part:BBa K1850000"

Latest revision as of 00:14, 24 September 2015

pRha - fimH

This part contains the fimH adhesin under control of a titratable rhamnose promoter BBa_K902065.

Usage and Biology

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 that bind to Nickel, Stainless Steel, and Caco2 colorectal cancer cells have been experimentally validated.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1850000 short</partinfo>
-This part contains the ''fimH'' adhesin under control of a titratable rhamnose promoter. The FimH protein is a subunit of a naturally occurring structure in some strains of ''E. coli'' called Type 1 Pili. These hairlike appendages ypically 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. 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.
+This part contains the ''fimH'' adhesin under control of a titratable rhamnose promoter [https://parts.igem.org/Part:BBa_K902065 BBa_K902065].
+===Usage and Biology===
+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.
-We established that our engineered system could control pili expression by performing an agglutination (link to first description and graphics in strains and assays). Following our protocol, we mixed cultures of our induced and uninduced plasmid-containing fimB knockouts with S. cervisiae yeast along with controls. We found that our plasmids recovered agglutination in the negative control strain and that this agglutination was dependent on the addition of inducer molecules. This shows both that we have control over expression of the biosynthetic machinery and the fimH adhesin, and that the resulting pili were functional as determined by a standard assay for mannose binding. This assay was performed in biological triplicates with the same result.
+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.
-===Usage and Biology===
+<html><body>
+<img src="https://static.igem.org/mediawiki/parts/f/fb/Flocculation_with_inducers.png" height="475px" width="750px"/>
+</body></html>
+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_K1850006 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:
+https://static.igem.org/mediawiki/2015/2/22/Harvard_Fim_Animated.gif
+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 that bind to Nickel, Stainless Steel, and Caco2 colorectal cancer cells have been experimentally validated.
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