To develop an oral cancer detection device, we noticed that the pH conditions in human biological samples are able to affect the expression of reporter protein through influencing folding processes and changing protein structures1,2. Consequently, while we were finding a suitable expression protein for our detection device, we tested the effect of different pH conditions on mRFP (BBa_E1010), a monomeric red fluorescent protein firstly characterized by Robert E. Campbell et al.. In this experiment, we measured and observed the excitation and emission spectra of mRFP under six pH level, and described the appropriate working and measuring conditions of mRFP.
We expressed mRFP through the addition of T7 terminator on an existing part - BBa_K199118 so that the new part (BBa_K3431048) can be expressed through our modified PURExpress in vitro protein synthesis kit (for further inquiries, please refer to our Measurement page). We reproduced the conditions of pH 2, pH 5, pH 7, pH8, pH 10, and pH12 by mixing different chemical compounds in double-distilled water. After expressing mRFP through the kit, we measured its excitation and emission spectra with a Synergy H1 microplate reader. We performed our data analysis and presentation through GraphPad Prism 5. For further interests, the procedures for this experiment can be found at our Protocol page.
From the two figures below, we observed that regardless of excitation and emission spectra, the fluorescent intensity of mRFP decreases in relation to the drop of pH levels (with the exception of pH 7 and 8 in excitation spectra). This represents that mRFP absorbs and emits light energy better under a more alkaline environment.
We calculated the average peak wavelength for excitation and emission spectra. The average peak wavelengths, which is 560 nm for excitation spectra and 609 nm for emission spectra, are quite dissimilar from the data on iGEM Registry of Biological Parts: the peak wavelength for excitation spectra is around 585 nm and 607 nm for emission spectra. The average peak wavelength in the excitation spectra is lower than that in the documentation on iGEM Registry of Biological Parts. The reasons can be attributed to a shift of maximum fluorescent intensity toward lower wavelengths under the condition of pH 12, indicating that a more alkaline environment can alter the excitation characteristics of mRFP. This suggests that a higher pH level makes mRFP more susceptible to be excited by a lower wavelength of a laser beam.
Through performing excitation and emission spectroscopy on mRFP under different pH levels, we concluded that mRFP performs better while shifting its peak excitation wavelength toward a lower number under alkaline conditions. Since the biological samples for oral cancer detection mainly consist of environments that are not overly alkaline3, this characterisation of mRFP is not particularly useful in our project. Nevertheless, the result of our experiment shows that mRFP is more suitable for observation in alkaline conditions with a lower excitation wavelength to reach the best emission result for this fluorescence protein.