Part:BBa_K4449011
Apt4UTI C3
Description
This composite part is formed by assembling the coding region of Bhp ( 6909 bp ) into the expression vector pET28b+, in frame with the T7 promoter and 6 X His affinity tag (5617 bp to 5634 bp ) of the expression vector.
Biology
This year team iGEM22_ IISER_ Berhampur has added a new part in the registry named Bhp (BBa_K4449002) . The insert (Bap-like Bhp ) part encodes a virulence factor of S. epidermidis that helps in biofilm accumulation and is known as a surface adhesion protein .It is commonly found in S. epidermidis strains.
Usage
This construct will be transfected into a host ( BL21DE3 ) and the protein production will be induced by the addition of IPTG. This protein will further be purified by the Ni NTA affinity chromatography ( due to the presence of the C terminal His tag ). In our project, this construct will help in the expression of this part under the control of the T7 promoter of this expression construct. We will express this protein with a C terminal 6X His tag in the BL21DE3 strain of E coli under IPTG induction and purify the protein using the Ni NTA affinity column. This part will be used for the negative selection round of ligand-based SELEX and will serve as a negative control in our ELISA-based binding assays. We have chosen this part from Staphylococcus epidermis because Staphylococcus epidermis comprises a minority of the microflora of the lower part of the Urethra, and can be expected to be present as a contaminant in the urine cultures.
Characterization
1. After purification, we will perform an SDS PAGE where we will run the molecular mass markers ( lane 1), Coomassie blue stained soluble cytoplasmic preparation( lane 2), the FimH1 His protein obtained by Ni NTA chromatography ( lane 3), and the solublecytoplasmic fraction after removal of the histidine-tagged FimH 2 protein (lane 4) and the purified Staphylococcal Bap like Bhp protein (lane5 – negative control).
2.Receptor blots -Upon running the purified sample on an SDS PAGE the samples will be transferred to PVDF microporous membrane filters using a semi-dry blotting apparatus.
3. Receptor blots of Staphylococcal Bap-like Bhp protein with His tag to α-D-mannosylated BSA (Sigma) will be carried out. In short, filter blots will be first incubated with α-D-mannosylated BSA (0.5 mg l-1) followed by incubation with rabbit anti-BSA serum, and finally with a peroxidase-conjugated anti-rabbit serum to demonstrate that Bap like Bhp protein shows no interaction with the receptors that are specific for Fim H. Hence we can validate the possibility of the Staphylococcus epidermis being more of a contaminant in urine samples and is non UTI causing pathogen. Moreover, we can compare and quantitate the concentration of Fim H1, Fim H2, and Bap-like Bhp protein by the measurement of the signal emitted by the protein bands. The signal intensity of the band is directly proportional to the concentration of the target protein.
4.Binding assays - His-tagged protein (Bap like Bhp 6X His) will be incubated with biotin-labeled anti-His antibody, which is immobilized to streptavidin-coated wells. Upon incubation of FITC-labeled aptamers with protein-coated wells, HRP-labeled anti-FITC antibodies will be added. After washing, QuantaBlu Fluorogenic Peroxidase Substrate will be added as a readout of the aptamer-non target binding and the fluorescence intensity is measured using a plate reader.
We would expect very low or no intensity of the emitted light as compared to the control.
For more details related to the assays visit our wiki page "https://2022.igem.wiki/iiser-berhampur"
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