Part:BBa_K5097016:Experience

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Applications of BBa_K5097016

E. coli DH5alpha cells expressing this construct were lysed by sonication, and the His-tagged sBFP was purified by IMAC. After buffer exchange and concentration, the purity of the protein product was visualized after SDS-PAGE and coomassie staining. (Figure 1). Experiments were performed to understand the impact pH would have on the fluorescence of their reporter system.

Figure 1: Coomasie stained PAGE gel showing purification of BFP-6XHis and RFP-6XHis

eBFP was loaded into a 96-well plate and buffer was added to a final volume of 100 µL, yielding a protein concentration of 0.566 ng/ml. The buffers used range from pH 4.0 to 9.0. 100μL of each buffer alone was added in another row, and fluorescence was then measured using Fluorescence Spectroscopy. An emission scan quantified by signal intensity runs from 400 nm to 500 nm with a fixed excitation wavelength of 374nm.

Figure 2: Graph comparing the maximum intensity emitted at each pH tested

Figure 3: Graph of the emission wavelength that produced the best intensity at each pH

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