Proof of concept:
The PBP-GFP was purified through Ni-chelating affinity chromatography. The target protein, PBP-GFP, is marked in the red box, has a molecular weight of 60kDa. As demonstrated in channel 1-4, protein is identified at the position of 60kDa, PBP-GFP is expressed in the E.coli; as demonstrated in channel 8-11 that the target protein is found in all 4 elutes, we have successfully purified PBP-GFP from the E.coli.
The target protein from channel 8-11 of electrophoresis figure 1 was purified through Q Sepharose Fast Flow. Proteins in well 3-5 have clear marks at 60kDa, indicating our construction can express the GFP-PBP we desire.
We incubate penicillin on the coated wells on the plate, add free penicillin (with varied concentration listed in Table 1) and PBP5-GFP, and allow them to react, during the process of which bound penicillin and free penicillin in the sample compete for PBP5-GFP, leading to the result that certain amount of PBP5-GFP would bind to free penicillin instead of the bounded penicillin. Testing the intensity of green fluorescence in the liquid sample, we are able to identify the amount of PBP-GFP, which also indicates the amount of free penicillin in the sample.
We conduct three different competitive binding experiments: the first two experiments enable us to determine one proper condition under which PBP works the best. And we use preprocessed milk sample in the third experiment, proving that our method can work under real life condition. The details and data are provided below.
The first competitive binding experiment:
In the first competitive binding experiment, we compare the result of applying different concentrations of penicillin. By calculating the correlation of each experiment, we discovered that 25 times diluted PBP-GFP5) leads to data with highest relativity. (See more in protocol)
The second competitive binding experiment:
The second experiment is conducted under the condition of 25 times diluted PBP5-GFP solution. By analyzing the data and applying the Pearson correlation coefficient, we are able to determine the excitation wavelength that ensure the highest relativity is be 405nm. (See more in protocol)