To test if our pH sensitive constructs express the fluorescence proteins we cultivated the bacteria hosting the plasmids in LPM with pH 7 overnight. For expression control for our alx-mNeonGreen construct, we inoculated 20 ml LPM with pH 7.0, 8.0 and 8.5 to an OD₆₀₀ of 0.2 using the overnight culture. After 20 and 40 minutes 1 ml of each culture where taken and adjusted to the lowest OD₆₀₀ of the three samples to standardize OD₆₀₀. This was necessary growth at pH 8.0 and 8.5 was significant slower than at pH 7.0. This diluted samples where used to measure fluorescence (extinction 490 nm, absorbance 520 nm) and again OD₆₀₀ with our plate reader. Three aliquots of each sample where measured (n=3), fluorescence was divided by OD ₆₀₀ and average of the three aliquots where taken to obtain the data shown in Figure 3.

As shown in figure 1. Expression of mNeonGreen is increased at pH 8 and 8.5 compared to standard pH 7. Cultures at pH 7, but also cultures without the mNeonGreen plasmid (data not shown) show a high basic fluorescence, leading to the conclusion that the cells naturally show emission at 520 nm. Still, fluorescence is increased twofold at pH 8.5 after 20 and 40 minutes and fluorescence is significantly increased at pH 8 at both measuring points