Part:BBa_K4449010
Apt4UTI C2
Description
This composite part is formed by assembling the coding region of FimH2 ( 534 bp ) by performing PCR amplification of the gene sequence derived from pBAD FimH 9X His(Addgene #97305), into the expression vector pET15b( Novagen, EMD Millipore), in frame with the T7 promoter and N terminal 6 X-His affinity tag of the expression vector.
Biology
This year team iGEM22_ IISER_ Berhampur has added a new part in the registry FimH2 BBa_K4449001.In this construct, the inserted basic part ( FimH2) 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, FimH 2 acts as a receptor-recognition element and hence as an adhesin that is responsible for the pathogenesis of urinary tract infection.
Usage
The construct will be used to express FimH containing N terminal His tag and with a thrombin site .This construct will be transfected into a host ( BL21DE3 ) and the protein production will be induced by the addition of IPTG. The expressed protein protein will further be purified by the Ni NTA affinity chromatography ( due to the presence of N terminal His tag ). Thereafter,the purified fusion construct containing the Fim H2 protein fused to the N terminal His tag will subsequently be tested with the selected candidate aptamers ( obtained from a later stage of ligand-based SELEX) for target binding, via ELISA-based binding assay. Due to the presence of a "Thrombin site" which acts as 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 will be used to cleave the 6X His affinity tag .After cleavage, the obtained Fim H2 protein will be 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 Fim H 2 protein and are showing no interaction with the His Tag.
By designing this construct we will be setting up a platform to purify tag-free Fim H protein.
Characterization
We will be using IPTG-based protein induction can be performed by adding an increasing concentration of IPTG and will purify the protein via Ni NTA column. So here we can estimate the trend of protein induction with the increasing concentration of IPTG.We will be also performing certain assays to test our parts like –
1. Validation and estimation of the concentration of the purified protein by running an SDS PAGE against molecular markers
2. Perform Receptor blots of FimH26XHis and FimH2 ( His tag cleaved) to α-D-mannosylated BSA to test receptor affinity of the purified protein.
3. Perform ELISA-based binding assays to find out the aptamer candidate showing the highest binding affinity towards the target.
For more details related to the assays visit our wiki page "https://2022.igem.wiki/iiser-berhampur"
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