Part:BBa_K1850010
pRha - fimH 136 KO - RPMrel - SpyTag_225 - HisTag_225
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 amine 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 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 added 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 engineered E. coli bound selectively Caco-2 cancer cells. Our control without RPMrel did not bind.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 765
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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