This circuit is designed for functional test of the RFP-FimH fusion protein. So we added the LacI regulated promoter in front of the coding sequence. For more information about RFP-FimH fusion protein, please check BBa_K1104101.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 781
Illegal AgeI site found at 893
- 1000COMPATIBLE WITH RFC
Literature survey- Contribution by Team IISER__Berhampur 2022
Group -Team IISER_Berhampur 2022
iGEM13_NYMU-Taipei devised a fluorescence-based methodology to test the functionality of the plac+RFP-FimH ( BBa_K1104102 ) protein by expressing a fusion protein of RFP-FimH and then checking the Fim H binding to mannose . From our literature surveys related to the FimH protein , we have found another methodology that can serve as a functionality check of purified FimH binding to mannose .The technique is termed as Receptor Blot.
1. fimH156 coding sequence was cloned ( encodes N terminal adhesive domain , amino acid residues 1-156) in plasmid pPKL241 ,and then transformed in HB101F’ cells . The FimH156-His protein was then produced upon induction with 1 mM IPTG for 1 hour.
2.The expressed FimH -6X His was purified from periplasmic fraction by Ni-NTA affinity chromatography.
3.Samples were then subjected to SDS^PAGE (15%) gel electrophoresis [ molecular mass markers ( lane 1), Coomassie blue stained of periplasmic fraction preparation( lane 2), FimH156- His protein after purification by Ni-NTA chromatography (lane 3)and the periplasmic fraction after removal of the histidine tagged FimH protein (lane4)]and transferred to PVDF microporous membrane filters using a semi-dry blotting apparatus.
4.Filter blots were first incubated with alpha-D-mannosylated BSA (0.5 mgl-1), followed by rabbit anti-BSA serum, and finally with peroxidase-conjugated anti-rabbit serum.
Although the utility of pLac+RFP-FimH part in testing the binding of FimH to mannose on E Coli is an excellent tool as conceptualized by Team iGEM13_NYMU-Taipei, we also explored the potential of expressing FimH with a histidine tag and using the Receptor Blot technique in testing the functionality of the FimH protein.
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