Team:NAWI Graz/Results

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Temperature Sensing

To test if the ibpa promoter actually promotes GFP expression dependent on cultivation temperature, we observed bacterial cultures harboring the expression vector at different temperatures over time. Diagram A in figure 1 shows the fluorescence intensities we measured all 15 minutes over 6 hours. Cultivation at 28°C lead to a very slow but steady increase in fluorescence intensity, whereas cultivation at 37°C resulted in a fast and steady increase in fluorescence intensity. For comparison of the different samples, we also divided the value for each sample’s fluorescence intensity by its respective OD₆₀₀(= relative fluorescence) to take the cell growth into account. The results are shown in diagram B in figure 1. When cultivated at 28°C, the relative fluorescence of the bacterial culture decreased throughout the whole experiment, down to a base level of fluorescence (blue line). When cultivated at 37°C, we measured a small decrease for the first hour and afterwards a strong increase of relative fluorescence with time (yellow line). When the temperature was changed to 37°C after three hours of incubation at 28°C, the relative fluorescence started to rise significantly within less than an hour (orange line). When lowered to 28°C from 37°C the intensity of relative fluorescence decreased (grey line).

Figure 1:  Change of fluorescence intensity during cultivation in four bacterial cultures. Cultures 1 and 2 were incubated at 28°C and cultures 3 and 4 at 37°C. After 3 hours (red line) the incubation temperature of culture 2 was increased to 37° and the temperature of culture 3 is decreased to 28°C. The excitation wavelength was 485/20 nm and the measured emission wavelength was 531/20 nm. A) The measured fluorescence intensity is depicted. B) Relative fluorescence, each measured fluorescence intensity was divided by the measured OD₆₀₀ of the sample.

The change in relative fluorescence with change of temperature indicates that the construct is responding to induction by increase in temperature. When incubated at 37°C, there was a strong increase in overall and relative fluorescence. This result confirms the induction of the ibpa promoter by heat. However, it was also shown that it took some time for the bacteria to adapt to the altered temperature, until a change in fluorescence could be observed. If the culture had already been incubated in the cultivation flask for 3 h at 28°C, a subsequent rise in temperature to 37°C led to a strong increase in fluorescence within less than 1 h. It must be noted though, that the cultivation flasks had to be taken out of the incubator for a short time every 15 minutes to take the samples for the measurement of fluorescence, which might have interfered with Gfp production. Moreover it could be shown that a decrease in temperature from 37°C to 28°C led to lower activity of the ibpa promoter and consequently the production of fluorescence protein decreased. The reduction of Gfp expression happened shortly after the temperature was reduced, within about 15 min. Although the Gfp production clearly dropped, when the cultures were incubated at 28°C, there was still a small level of expression. In relation to the cell density measured by OD₆₀₀, the fluorescence significantly decreased. However, the 3 h cultivation time at 28°C was not enough to reduce either fluorescence intensity or relative fluorescence to the level of the culture cultivated at 28°C over the whole experiment.

It has to be noted that the ibpA promoter is already active at a temperature of 37°C. This corresponds to the temperature optimum of E. coli and may come unexpected for a heat shock promoter. However, when the bacterial culture is grown at a lower temperature like 28°C, the increase to 37°C can be used as a heat shock for induction. To further shorten the time span needed for induction of the ibpa promoter, it would be beneficial to test the expression construct at even higher temperatures. In this case, it would be important to keep an eye on cell growth as E. coli does not tolerate growing under extreme heat shock conditions for an unlimited amount of time. Additionally, it might be possible to further improve the promoter in the future via site-directed mutagenesis.

pH-Sensing

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 20ml 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 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 OD₆₀₀ and average of the three aliquots where taken to obtain the data shown in Figure 2.

Figure 2. Alx controlled mNeonGreen Expression: Culture media was bufferd to pH 7, 8 and 8.5 and inoculated to an OD₆₀₀ of 0.2. After 20 and 40 minutes, aliquotes where taken and fluorescence (extinction 490 nm, absorbance 520 nm) and OD₆₀₀ were measured with the plate reader (n=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.