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 of 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 1ml of each culture was taken and adjusted to the lowest OD₆₀₀ of the three samples to standardize the OD₆₀₀. This was necessary as growth at pH 8.0 and 8.5 was significant slower than at pH 7.0. The diluted samples were used to measure fluorescence (excitation 490 nm, emission 520 nm) and again OD₆₀₀ with our plate reader. Three aliquots of each sample were measured (n=3), fluorescence was divided by OD₆₀₀ and averages of the three aliquots were taken to obtain the data shown in Figure 1.

As shown in Fig. 1 fluorescence increases at pH 8.0 and 8.5 at both times compared to a standard culture at pH 7.0. At pH 7.0 cultures still show high fluorescence values with no difference between cultures with our plasmid or cultures without any DNA coding for mNeonGreen (Data not shown). This leads to the conclusion that bacteria on their own emit at ~520 nm when stimulated at 490 nm. Still fluorescence is significantly increased in all cultures at higher pH with a maximum of fluorescence at pH 8.5 after 20 min. This increase in fluorescence allows to distinguish between induced and uninduced. Hence we could show that our construct works as planned. The next step would be to test how much base (NaOH) would be needed to change the pH of a defined volume of culture with pH 7.0 to pH 8.5. This data would enable us to use the construct to control the robot. Unfortunately the summer was to short to complete this task.