Difference between revisions of "Team:NAWI Graz/Results"
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| − | Figure 1: Change of fluorescence intensity during cultivation in four bacterial cultures. Cultures 1 and 2 were incubated | + | <font size="-2"><b>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 | 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 | 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. | 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. | + | B) Relative fluorescence, each measured fluorescence intensity was divided by the measured OD600 of the sample.</font> |
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| − | <p>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.</p> | + | <p> |
| + | <br> | ||
| + | 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.</p> | ||
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Revision as of 12:28, 31 October 2017
RESULTS
Temperature Sensing
To test if the ibpa promoter actually promotes GFP expression dependent on the temperature, we observed bacterial cultures
harboring the expression vector at different temperatures over time. Diagram A in figure 1 shows the fluorescence
intensity we measured all 15 minutes over 6 hours. Cultivation at 28°C leads to a very slow but steady increase
in fluorescence intensity, whereas cultivation at 37°C results 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. The bacterial culture showed a decrease in relative fluorescence, when cultivated
at 28°C 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 (blue line). When lowered to 28°C
from 37°C the intensity of relative fluorescence decreases (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 hours at 28°C, a subsequent rise in
temperature to 37°C led to a strong increase in fluorescence within less than an hour. It must be noted though,
that the cultivation flasks had to be taken out of the incubator for a short amount of time every 15 minutes
to take the samples for the measurement of the fluorescence, which might have interfered with GFP production.
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 especially important to keep an eye
on cell growth as E. coli wouldn’t tolerate growing under extreme heat shock conditions for an unlimited amount
of time. Additionally, in some circumstances it might be possible to further improve the promoter in the future
via site-directed mutagenesis. Moreover it could be shown that a decrease in temperature from 37°C to 28°C led
to inhibition of the ibpa promoter and consequently the production of fluorescence protein nearly stopped. The
reduction of GFP expression happened already shortly after the temperature was reduced, within about 15 minutes.
Although the GFP production clearly dropped, when the cultures were incubated at 28°C, there was still a small
level of expression. However, in relation to the cell density measured by OD600, the fluorescence significantly
decreased. After all, this relative fluorescence acted as the signal that was detected and processed by the associated
communication devices.
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.
