Part:BBa_K4449009
Apt4UTI C1
Description
This composite part is formed by assembling the coding region of FimH1 ( 540 bp ) gene whose sequence is derived from the pBAD FimH 9X His plasmid(( Addgene #97305)), into the expression vector pET28b+(EMD Biosciences), in frame with the T7 promoter and C 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 FimH1(BBa_K4449000).Here ,the DNA part or the insert contains the coding sequence of the E.coli UTI89 FimH1 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 1 acts as a receptor-recognition element and hence as an adhesin that is responsible for the pathogenesis of urinary tract infection.
Usage
Fim H1device which is cloned in this plasmid construct is regulated by the lac repressor that binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
This construct will be transfected into a host ( BL21DE3 ) and the protein production will be induced by the addition of IPTG. This fusion protein ( Fim H1 -6X His) will further be purified by the Ni NTA affinity chromatography ( due to the presence of C terminal His tag ) and will be tested for the binding affinity towards our aptamer candidates. The DNA part or the insert contains the coding sequence of the E.coli UTI89 FimH1 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 1 acts as a receptor-recognition element and hence as an adhesin that is responsible for the pathogenesis of urinary tract infection.
Design consideration
Here Fim H1 will be cloned between the Nco1 and Xho 1, to get rid of the N terminal His tag, thrombin site, and the T7 tag and retain the C terminal His tag with the expressed Fim H1 protein. This is because the C terminal His tag allows proper protein folding and this will ensure non interference of our aptamer binding to the thrombin site and T7 tag while performing binding assays.
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 the 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 FimH1-6XHis 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 assay visit our wiki page "https://2022.igem.wiki/iiser-berhampur"
References
1. Schembri, M.A., Hasman, H., and Klemm, P. (2000). Expression and purification of the mannose recognition domain of the FimH adhesin. FEMS Microbiology Letters, 188(2), pp.147–151. doi:10.1111/j.1574-6968.2000.tb09186.x.
2. Tao Wang, Wang Yin, Hadi AlShamaileh, Yumei Zhang, Phuong Ha-Lien Tran, Tuong Ngoc-Gia Nguyen, Yong Li, Kuisheng Chen, Miaomiao Sun, Yingchun Hou, Weihong Zhang, Qingxia Zhao, Changying Chen, Pei-Zhuo Zhang, and Wei Duan, 2019 Feb;30(1), A detailed protein-SELEX protocol allowing visual assessments of individual steps for high success rate, DOI: 10.1089/hgtb.2018.237, Human Gene therapy
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3738
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 4074
Illegal PstI site found at 3738 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3738
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3738
Illegal NgoMIV site found at 126
Illegal AgeI site found at 3896 - 1000COMPATIBLE WITH RFC[1000]
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