Part:BBa_K3953004
fimH HIS Tag
The histidine tag sequence that codes for consecutive histidine amino acid residues within the fimH gene. The affinity of histidine tags for NiNTA beads will serve as a proof of concept for fimH binding.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 872
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 872
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 171
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 872
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 872
- 1000COMPATIBLE WITH RFC[1000]
Literature survey and improvement - Contribution by Team IISER_Berhampur 2022
Group - Team IISER_Berhampur_2022
Summary 2022
fimH HIS Tag is a DNA part that was developed by iGEM21_Pittsburgh (2021-10-21) which consists of the histidine tag sequence that codes for consecutive histidine amino acid residues within the fimH gene.
Although this is an excellent tool in which the affinity of histidine tags for NiNTA beads will serve as a proof of concept for fimH binding , from literature surveys we found that an N or a C terminal His tag is more desirable for NiNTA based purification.[1]
In our project Apt4UTI , we have developed a platform to express C terminal and N terminal His tagged FimH protein, since we through our literature survey we learned that putting a His tag at C terminal or N terminal end are usually desired approach [3] without interfering with the protein conformation and will ease purification process.The C terminal and N terminal His tag would enable proper folding of our protein of interest FimH1 and FimH2 . FimH1 and FimH2 are the modified versions of the gene sequences derived from the plasmid pBAD-Fim H -9x His ( Addgene # 97305) which were developed for cloning into pET28b+ ( EMD Biosciences)and pET15b+( Novagen ,EMD Millipore) respectively.
Apt4UTIC1 ( BBa_K4449009) will express our insert FimH1 with C terminal His tag
Apt4UTIC2 ( BBa_K4449010) will express our insert FimH2 with a N terminal His tag.
In the construct Apt4UTI C2 , the FimH2 will be expressed with a N terminal His tag along with a thrombin site , so that the enzyme thrombin can be used to remove the His tag in order to purify a tag free protein and validate that the aptamers are not showing any non-specific binding with the His tag .
Methodology
With the help of our literature surveys we have devised several methodologies to measure our parts
Methodologies to test our construct Apt4UTIC1 which will express C terminal 6X His tagged FimH1.
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.ELISA based Binding assays - His-tagged protein ( Fim H1 -6XHis ) will be incubated with a biotin-labelled anti-His antibody, which will be immobilized in streptavidin-coated wells. After incubation of FITC-labelled aptamers with protein-coated wells, HRPlabelled anti-FITC antibodies will be added. After washing, QuantaBlu Fluorogenic Peroxidase Substrate will be added as a readout of the aptamer-target binding and the fluorescence intensity is measured using a plate reader[2].We would select those aptamer candidates which shows high intensity fluorescence signal upon interacting with Fim H1 and no or low signal intensity on interacting with Bhp .On the other hand we need to rule out those aptamers which emits positive signals upon interacting with Bhp .
Methodologies to test our construct Apt4UTIC2 which will express N terminal 6X His tagged FimH2.
1. Validation and estimation of the concentration of the purified protein by running an SDS PAGE against molecular markers
2. Perform Receptor blots of FimH2-6XHis to α-D-mannosylated BSA to test receptor affinity of the purified protein.
3.ELISA based Binding assays- His-tagged protein ( FimH2 -6XHis ) will be incubated with a biotin-labelled anti-His antibody, which will be immobilized in streptavidin-coated wells. After incubation of FITC-labelled aptamers with protein-coated wells, HRP labelled anti-FITC antibodies will be added[2]After washing, QuantaBlu Fluorogenic Peroxidase Substrate will be added as a readout of the aptamer-target binding and the fluorescence intensity is measured using a plate reader[2]We would select those aptamer candidates which shows high intensity fluorescence signal upon interacting with FimH2 and no or low signal intensity on interacting with Bhp .On the other hand we need to rule out those aptamers which emits positive signals upon interacting with Bhp.
References
1.Schembri, M.A., Hasman, H. and Klemm, P; Expression and purification of the mannose recognition domain of the FimH adhesin;01 July 2000; 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; A detailed protein-SELEX protocol allowing visual assessments of individual steps for high success rate, 2019 Feb30, Human Gene Therapy MethodsVol. 30, DOI: 10.1089/hgtb.2018.237
3. His tagged Proteins ; Creative biostrucrure https://www.creative-biostructure.com/
For more information related to our project visit "https://2022.igem.wiki/iiser-berhampur"
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