Buffer Assays

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.

Developing pH media

Next, we need to determine how much buffer is required to stabilize the pH. To begin, we used biologically friendly buffers, including citrate, 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), and 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris). However, the problem is that since the bacteria is continually growing in the overnight culture, it would continue to produce waste product, acetate, that will lower the pH. The amount of buffer that would be needed to neutralize the acetate would prove to be detrimental to the growth of the bacteria even for biologically friendly buffers. As a result, we looked for the highest amount of buffer that the cell can tolerate.

From our experiments, pH 3.5 did not grow at all evident in the low levels of OD across all buffering concentrations. This set of data showed that the cells can tolerate 100 mM of buffer without affecting growth. However, the pH is still changing dramatically. The only final pH that stayed within 0.5 pH is for pH = 3.5 and 4.5. However, this is most likely due to the low optical densities. With less cells, less buffer is needed to balance the waste product. In the next experiment, we tried to determine the upper limit that cells can tolerate.

The data set do not include pH = 3.5 because the pH is not conducive for growth for our strain since nothing has grown in it. As a result, it does not matter the buffering range. This experiment is somewhat successful in finding an upper limit. The optical densities for both pH 5.5 and 6.5 trend down as the concentration of buffer increases. Tris at 250 mM appers to be toxic for the cells. At the same time, the buffering capacity was high enough to allow for a final pH fluctuation of within 0.5 . Knowing this, we chose the following buffering concentrations.

pH = 3.5, 50 mM

pH = 4.5, 50 mM

pH = 5.5, 100 mM

pH = 6.5, 150 mM

pH = 7.5, 150 mM

Fluorescence Results