Pre-Fluorescence Assay Work

We have chosen to measure fluorescence level as a proxy for promoter acitivity under different pH conditions. The primary concern for the project is the promoter’s sensitivity to acidic conditions. Since low pH is the ultimate cause for dental cavities, our construct is designed to turn on under acidic conditions. To do this, we have placed green fluorescence protein (GFP) downstream of each of our promoter, and to make sure the media will not interfere with the cells’ fluorescence, we used clear minimal growth media (M9).

M9 Media’s Buffering Capacity

First, we need to determine whether M9 media is capable of buffering the solution at an adequate level so that the pH of the overnight culture does not change by more than 0.5 pH. Since M9 contain different phosphate compounds, it acts as a relatively good buffer, especially at the range of pH 6-8. To test this, we grew our constructs in the following pH: 3.5, 4.5, 5.5, 6.5, and 7.5. The pH is adjusted by adding HCl and NaOH solution to the M9 solution. The results that pH 3.6 is not conducive to cell growth due to the low optical density. For the other pH ranges, the pH changed by more than 1. Our results show that a buffer is needed to stabilize the pH.