Part:BBa_K4449001
FimH2
The DNA part contains the coding sequence of the E.coli UTI89 FimH2 signal peptide and lectin domain which forms a subunit of Type 1 fimbriae, located at the tip of the organelle as an integral part of short fimbriae. These structural organelles help the Uropathogens to mediate specific adhesion to alpha -D-mannoside receptors of the host and represent hair-like structures on the surface of E.coli. Therefore, FimH2 will act as a receptor-recognition element and hence as an adhesin that is responsible for the pathogenesis of urinary tract infection. This part is retrieved from the plasmid pBAD - FimH -9X His that is deposited in the Addgene repository by Per Svenningsen Lab.
This part will be first expressed as a fusion protein with the N terminal fusion tag in the pET15b expression vector and will subsequently be tested for binding with e selected candidate aptamers ( obtained from a later stage of ligand-based SELEX) via ELISA-based binding assay. Here the plasmid contains a "Thrombin site" which refers to a DNA sequence which contains the recognition site of thrombin ( a protease). As a control experiment, after purification of the induced FimH2 protein carrying 6 X His affinity tag at its N-terminal end, thrombin enzyme will be used to cleave the affinity tag and again tested with the selected candidate aptamers ( obtained from a later stage of ligand-based SELEX) to validate if the aptamers are truly binding to the purified FimH2 protein and is showing no interaction with the His Tag. Thus by engineering this part we will be setting up a platform to purify His tag-free protein.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 232
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 232
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 232
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 232
Illegal AgeI site found at 390 - 1000COMPATIBLE WITH RFC[1000]
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