Soil Survey Results
An investigation into potential diazotroph presence was conducted to gain a better insight into the current impacts of nitrogen fixation in the soils of varying local regions. By surveying and testing 12 different soil samples from differing geographical locations, biological activity, human impact, and composition, and the effect of different bacteria in these soils, we were able to conclude that diazotrophs not only were a viable solution that could be implemented to local Australian soil; furthermore it was highly likely that species of diazotrophs were already present, and fixating nitrogen already in some of our soil.
This experiment observed the changing pH of a range of media that were inoculated with bacteria from the wide variety of soil samples. These media consisted of semi-solid agar, agar plates, and liquid broths containing bromothymol blue indicator to visualise the different survivability and prevalence of such bacteria in glucose, sucrose, malate, and mannitol sources as media.
The media turning from green to blue indicated that it became more basic, potentially due to the production of ammonia. Hence, a sample that turned blue hinted at the likelihood of nitrogen fixation occurring. Contrastingly, a change in colour to yellow shows the samples were turning more acidic, signifying the fermentation and growth of bacteria, as this would result in a release of carbon dioxide gas which would in turn increase the acidity of the sample.
GFP Fluorescence Results
Qualitative Testing
Handheld UV testing, a UV light was used to check for GFP fluorescence on the Agar plate which we incubated our assembled Prototypes. Fluorescence would indicate that our prototype worked and allowed us to advance in more detailed testing.
Then E coli is incubated into liquid culture at set desired atmospheric oxygen levels of anaerobic, 0.5% oxygen, 2% oxygen and aerobic conditions. Once cells are given time to incubate, bacteria growth is measured using a microplate reader. Then the fluorescence intensity of GFP at each condition will be recorded. Finally, analysis will be done by adjusting the relative intensity per bacteria growth and a graph will be generated to allow for further learning.
The test result will first allow us to determine whether our prototype is “oxygen sensitive” and secondly to determine which prototype variant ( A, B, C ) is best, with the most focused response and the largest range.
Methods
Preparing liquid construct
For each oxygen level, pour 20ml of set liquid construct (eg prototype A) into 50 tubes. Then repeat for the other two prototypes ( B and C). Then pour 20 ml of liquid nutrient without construct as control. In total, there should be 7 tubes of culture for each oxygen level and 28 in total.
Preparing set oxygen levels
Anaerobic conditions
To achieve anaerobic conditions, test tubes are placed within a sealed container with a hand warmer acting as a catalyst to use up the oxygen present in the container. Then the container is placed on a shaker set at 37*C and 90 rpm.
0.5% oxygen conditions
To achieve 0.5% oxygen conditions, a vacuum desiccator was first filled with Nitrogen to purge all oxygen out of the container. Then 5 ml of oxygen is introduced through the tap. Then a gas chromatography test is run to verify the percentage of oxygen. Then the desiccator is seal-clamped and placed on a shaker set at 37*C and 90 rpm.
2% oxygen conditions
To achieve 2% oxygen conditions, a vacuum desiccator was first filled with Nitrogen to purge all oxygen out of the container. Then 29 ml of oxygen is introduced through the tap. Then a gas chromatography test is run to verify the percentage of oxygen. Then the desiccator is seal-clamped and placed on a shaker set at 37*C and 90 rpm.
Aerobic conditions (16% oxygen)
Test tubes are placed on a test tube holder on a shaker set at 37*C and 90 rpm.
Loading Construct in microplate for imaging
Two biological replicates were grown overnight, and 150 µL of each culture was put into a 96-well plate in triplicate, and the plate’s OD600 was measured. The GFP fluorescence (485 nm Excitation, 520 nm Emission) was also measured in a BMG-Pherastar plate reader at a gain setting of 400. By imagining the samples, the bacteria growth of each sample could be quantified which would be useful for understanding the GFP fluorescence relative to the amount of bacteria present.
Measuring GFP fluorescence
GFP fluorescence data is recorded using a plate reader, 2 Biological replicants were taken for each Prototype and technical replicates for each value.
Quntitative Results
**The values of the blank were subtracted from the raw data, and normalised for OD600 measurement.
Graphing Results
Data collected above is generated into a graph to allow better understanding. The GFP fluorescence is adjusted according to bacteria growth per optical density at 600.The GFP intensity scale is normalised to 0 according to our Control, due to background fluorescence.
** Promotor A was taken out due to its abnormal reaction that we cannot explain at this point
** We will do research and re-experiment with promoter A in the future
Thus our experiment shows that our test cycle succeeded as FNR sensitivity of oxygen is proved to be successful as an oxygen sensor with promoters B (NarK) and C (DmsA) whereas it showed no O2 sensitivity in promoter A (NarG). As shown on the graph, high fluorescence intensity was observed when our prototypes were incubated in oxygen conditions below 2%. For the purposes of our project as a whole, we’ve determined that promoter B of NarK is most suitable for the continuation of our project as it exhibited more stable, consistent fluorescence across different oxygen intervals with a more compact spread of results than promoter C (DmsA) between the biological replicates.
As seen in the graph above, there is an increase in GFP production in both successful promoters from 0% to 0.5%. Also, both successful prototypes became inactive at ~18% oxygen approaching atmospheric conditions as expected. Hence, both systems successfully function as an oxygen sensor to the requirements of our experiment.
Further Prototype B Data
Future Plans
Following the success of FNR oxygen sensitivity, our next step is to substitute the sfGFP indicator with NifA, which regulates nitrogenase production. We believe that this will maximise the efficiency of nitrogenase production, by optimising its production to only occur when nitrogenase can function, under the ideal oxygen concentration levels. Through maximising the production of nitrogenase, we should be able to efficiently produce ammonia within the soil, effectively revitalising the ammonia content of the soil in a self-sustaining manner.
After incorporating the NifA regulator, future efforts could focus on developing a sensor to other soil factors, such as carbon-source availability, and concentration of minerals such as Iron and Molybdenum that are required in the process of nitrogen fixation. By successfully optimising our E.coli to be able to sense a multitude of environmental conditions, the efficiency of nitrogen fixation can be greatly enhanced.
