Part:BBa_M50440:Experience
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Applications of BBa_M50440
This construct can successfully be used to measure intracellular pH given by a BRET ratio in S. cerevisiae. Due to the well-characterized pH-sensitivity of Rluc8 (luciferase), we are confident that BRET readout from cpVenus (fluorophore) is indicative of intracellular pH because it was excited through Rluc8 activity.
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Yeast Entrainment and pH measurement
Methods
To entrain yeast to exhibit a circadian rhythm, we incubated our cells in a water bath system that changed temperature every 12 hours, oscillating between 21° C and 28° C. This was accomplished with a standard water bath and an outlet timer to turn the bath on or off every 12 hours. Fortunately, the water temperature settled at 21° C when the bath was turned off. We suspended the cells in 2% galactose SD-Leu media. Prior research has shown that this method is effective in entraining a circadian rhythm in yeast after two weeks of entrainment, which is expressed by a regular oscillation of media pH.2 This research was the basis of our project, as we worked under the assumption that the cytosolic pH would also oscillate in the presence of a 24-hour regular signal. We expected the cytosolic pH to reflect these changes in the media pH because the major contributors to these changes are glutamine synthesis byproducts, nitrogen metabolism, and the periodic transport of various amino acids and ammonium into the cell.2, After the two weeks had passed to entrain the yeast, we used a fluorometer (Agilent Cary Eclipse Spectrophotometer) to read one sample at a time, attempting to trace out enough time points to determine a circadian pattern within the yeast. We originally tried to use the plate reader to capture many duplicates of this experiment at once, but the emission signal was too low for the machine to detect, likely due to the very small volume of cell in each well. We eventually decided the fluorometer would be more accommodating to our plasmid construct’s output. Over a 42-hour period, we were able to collect data from the bioluminescence of our construct. We were unable to take readings from after 10pm to 8am, so we planned on taking data from available timepoints and extrapolating any patterns to the hours of the night. The protocol for data collection at these various time points during the day, shown in Figure 2, required a 50 mM KCl, 50 mM NaCl, and 50 mM HEPES buffer. For this type of experiment, an OD600 between 0.4 and 0.8. is typically used.1 For this quantity of cell, we found it was necessary to create a 10 μM coelenterazine solution. Once the cells were suspended in the HEPES buffer and the coelenterazine had been added, readings were automatically collected once a minute for anywhere from 15-40 minute at no excitation, 400-600 nm collection, and 20 nm slit. As this device is sensing the passage of time through pH and depends on a continuously changing variable instead of a specific value, it is very difficult to develop true positive and negative controls. While we could not formulate a method to develop a positive control, as a negative control, we collected BRET data from a sample of cells without the addition of coelenterazine. This provides a negative control, because without the substrate to be cleaved, we expect to see no light emission.
Results
After periodically taking readings over the course of forty-two hours, we ended with eight valid readings. The BRET ratio for each time point was calculated by dividing the average intensities of all 15-40 readings at 525 nm by that of at 475 nm. The BRET ratios against time to show changes in BRET, thus pH, over time.
Figure 2: Table showing the average light intensities (a.u.) at emission wavelength 525 nm and 475 nm, and the resulting BRET ratio for each time point.
In the Zhang 2012 paper, light intensity at 475 nm ranges from close to 0 to 3.5 a.u., and light intensity at 525 nm ranges from slightly below 1 to over 5 a.u, with a resulting BRET ratio of around 0.5 to 2.5, depending on the pH. Our results differ in that our emission values are much lower that of the paper, with every single average being lower than 1 a.u.. A negative light intensity would be considered invalid, but because the average is calculated from multiple different numbers and the ratio itself matters the most, we decided to include this as a data point. Also notably, at hour 14, the data resulted in a negative BRET ratio, which is also theoretically impossible. We also decided to include hour 14 as a datapoint, because these results could be simply interpreted as a very small number divided by a larger number.
Figure 3.1, 3.2: Intensities of the fluorophore at different wavelengths in our experimental sample of entrained yeast (10 readings overlaid) are compared to the negative control (1 reading) which does not have coelenterazine. These readings are from a single time point, and the average intensities at 525 nm and 475 nm will be used to calculate BRET ratio.
For each time point, the graphs for each reading were plotted on top of each other to form one composite graph. All of our bioluminescence graphs looked very similar, with a noticeable small peak around 425 nm, but no distinguishable peak around 525 nm. In comparison to our negative control, these experimental results are drastically different. The control sample shows bioluminescent intensity centered around zero for every wavelength, suggesting that no light is being emitted from the sample without coelenterazine. Our genetic construct is clearly producing the luciferase the reacts with the coelenterazine to emit light. Furthermore, observing a peak around 425 nm is further evidence that the pHlash construct is specifically suited for determining the BRET ratio.
Figure 4.1, 4.2: The same data is used to plot these two graphs, and are placed adjacent to each other for comparison. The first graph shows the BRET ratios from each day separately, fit to a curve, placed on the same graph with the x-axis scale from 6:30 am to 10:30 pm. The second graph shows the BRET ratios, fit to a curve, placed as continuous time points over the course of 42 hours.
We then graphed the BRET ratios at each timepoint to notice any patterns in pH throughout the forty-two hours. The ideal result is that we would observe a consistent wave pattern, with an entire cycle (a peak and trough) completing in 24 hours. Indeed, from Figure 4.2, we can observe maxima and minima occurring at approximately 24-hour increments.
Stanford Location
Plasmid name: pHlash
DNA2.0 Gene #: pD1204-NSH
Organism: S. cerevisiae
Device type: Sensor
Glycerol stock barcode: 133021752
Box label: BioE44 S18