Difference between revisions of "Part:BBa K1850010"

Revision as of 23:31, 18 September 2015

pRha - fimH 136 KO - RPMrel - SpyTag_225 - HisTag_225

Usage and Biology

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 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.

We wanted to take the E. coli that naturally colonize the gut and engineer them to specifically bind to cancer cells. We were able to locate a 1 amino acid substitution at site 136 on fimH that had been shown disrupt mannose binding via standard assays. We introduced these modifications separately to our fimH-HisTag plasmid (BBa_K1850006 via site-directed mutagenesis, including the His Tag for reliable measurement. This set us up to to test our hypothesis that these mutations would disrupt mannose-mediated agglutination of yeast cells. We then created this part by adding a peptide called RPMrel that had been shown to bind selectively to colon cancer cells. We conducted a dot blot assay and found that our part bound selectively to Caco-2 cancer cells, while our control without RPMrel (BBa_K1850008) did not bind.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 765
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 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 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 wanted to take the ''E. coli'' that naturally colonize the gut and engineer them to specifically bind to cancer cells. We were able to locate a 1 amino acid substitution at site 136 on ''fimH'' that had been shown disrupt mannose binding via standard assays. We introduced these modifications separately to our fimH-HisTag plasmid (<partinfo> BBa_K1850006 </partinfo> via site-directed mutagenesis, including the His Tag for reliable measurement. This set us up to to test our hypothesis that these mutations would disrupt mannose-mediated agglutination of yeast cells. We then created this part by adding a peptide called RPMrel that had been shown to bind selectively to colon cancer cells. We conducted a dot blot assay and found that our part bound selectively to Caco-2 cancer cells, while our control without RPMrel (<partinfo> BBa_K1850008 </partinfo>) did not bind.
+We wanted to take the ''E. coli'' that naturally colonize the gut and engineer them to specifically bind to cancer cells. We were able to locate a 1 amino acid substitution at site 136 on ''fimH'' that had been shown disrupt mannose binding via standard assays. We introduced these modifications separately to our fimH-HisTag plasmid (<partinfo>BBa_K1850006</partinfo> via site-directed mutagenesis, including the His Tag for reliable measurement. This set us up to to test our hypothesis that these mutations would disrupt mannose-mediated agglutination of yeast cells. We then created this part by adding a peptide called RPMrel that had been shown to bind selectively to colon cancer cells. We conducted a dot blot assay and found that our part bound selectively to Caco-2 cancer cells, while our control without RPMrel (<partinfo>BBa_K1850008</partinfo>) did not bind.
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