Part:BBa_K1850010
pRha - fimH 136 KO - RPMrel - SpyTag_225 - HisTag_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_K1850010 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. In this part, fimH contains a 6xHistidine Tag inserted at the 225 location shown above, along with a SpyTag peptide which should bind to SpyCatcher but has not been characterized (Zakeri et. al 2012). The 225 site was chosen for a HisTag modification to allow for functional inserts to the N-terminus (see above) while maintaining the versatility of a HisTag for measurement and protein purification.
A HisTag is string of repeated charged residues that has the ability to bind to nickel because of its charge, can be used for an antibody target in a Western blot, and can allow for rapid purification of chimeric fimH using Nickel- NTA bead purification. We were first interested in characterizing the utility of the HisTag as a target for detecting recombinant fimH and as a measurement platform for future teams using this part.
We performed an α-His Western Blot to characterize this element. Cultures of our His Tagged-fimH plasmid-containing pili knockout bacteria were OD standardized and separated into two subcultures, one of which was induced with Rhamnose, the other uninduced. Samples taken at three timepoints show increasing expression of fimH in the induced culture but not the uninduced. This demonstrates that we can express recombinant fimH and that the His Tag gives us traction to measure expression:
In principle, the HisTag on fimH could also be used to quantify recombinant expression in assays like whole-cell ELISA, both integrated with the pilus shaft and separately. In the future we hope to test the efficacy of these assays for determining quantitative expression levels. Additionally, the His Tag will allow future teams to purify recombinant pili using Nickel NTA purification for use in vitro.
We hypothesized the HisTag might also have functional utility binding to nickel. We used a nickel agglutination assay to detect whether our bacteria could bind to Nickel-NTA magnetic microbeads and aggregate them or remain in suspension. OD standardized volumes of induced and uninduced cultures were added to an equivalent volume of suspended Nickel-NTA beads, agitated, and allowed to rest. We found that our HisTag fimH plasmids were able to agglutinate nickel beads, pulling them out of suspension, while a Wild Type control was not (visit our [http://2015.igem.org/Team:Harvard_BioDesign/Metal wiki] for more assay information). This suggests control over adhesion to Nickel-NTA magnetic beads:
The ability to remove nickel particles from a solution could be extremely useful in waste water purification and pollution remediation. This experimental setup is almost identical to how we imagine our nickel binding system could be used in the real world. A sample of our E. coli could be added to a contaminated sample of water, the sample could be agitated, and any heavy-metal contaminants would fall out of solution with the bacterial clump. Future work can elaborate the toolbox of contaminant-binding peptides so that a range of harmful products could be purified. Furthermore, this process might just as easily be applied to mining valuable minerals from water, where binding peptides for rare and precious materials could be added to fimH, the resulting induced cultures mixed with a water sample containing the material of interest, and the bacterial clump extracted as if “mined” out of water. Our biological approach could be superior to existing mining techniques which require toxic chemicals and generate environmentally hazardous waste products. With biological extraction, the only waste would be a completely biodegradable and present in the water anyway (bacteria are present everywhere). These directions are areas of ongoing research in the lab.
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 this modification 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 this part bound selectively to Caco-2 cancer cells, while our control without RPMrel (BBa_K1850008) 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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