Team:RHIT/Results
Results
Results of the Fluorescence Measurements
Initially, experiments were carried out to determine if the riboswitches responded properly within our specific cell environment and with our construct. To determine if the riboswitches responded to the addition of the vitamin in the form of leucovorin calcium for the B9 riboswitches or cyanocobalamin for the B12 riboswitches, we plotted the normalized fluorescence against time. This produced plots like following:
Figure 1: Aliquots of the T. tengcongensis riboswitch culture were taken at 0,2,4,8, and 20 hours. The cultures were inoculated at 0 hours into either a solution of M9 minimal or M9 minimal and 0.03 mM of added cyanocobalamin.
Generally after 4-8 hours, a significant difference in GFP fluorescence can be observed between the assays with no added ligand and those with the ligand. By the 20 hour point, the cells show reduced GFP expression in both assays, though the expression levels between the two samples remains distinctly different. This reduction in expression per OD630 units is likely due to cell death within the liquid cultures. Because the most distinct difference between the riboswitches occurred 8 hours after the addition of the ligand, we plotted the normalized fluorescence against the concentration of the added ligand in the culture at this time, producing the following plots:
Figure 2: Vitamin B9 riboswitch results. All measurements were taken 8 hours after inoculation. The fluorescence excitation/emission wavelengths were 485 and 530 nm. OD630 measurements were taken at the same time. The fluorescence was adjusted to a negative control, and the OD630 was adjusted to the blank media.
The B9 riboswitches show variable fluorescence expression with no and low concentration of the ligand; however after the concentration of leucovorin calcium increases to and beyond 0.5 mM, the fluorescence approaches zero comparatively. The T. tengcongensis B12 riboswitch has the same trend when the concentration of cyanocobalamin increased, but the E. coli riboswitch begins with a much lower fluorescence output. Additionally, the T. tengcongensis B12 riboswitch is potentially slightly more sensitive, as a much more distinct difference is seen in the 0.03 mM concentration.
Figure 3: B12 riboswitch results. At 8 hours after inoculation, the fluorescence at an excitation wavelength of 485 and emission of 530 nm and OD630 measurements were taken. The fluorescence was adjusted to a negative control, and the OD630 was adjusted to the blank media.
This allows us to see distinct differences in output at different concentrations of the binding repressor and verify the operation of the riboswitches in the constructed parts.
Isothermal Titration Calorimetry Measurements
Figure 4: ITC graph using 20 2.49 µL injections of 1.23 M Vitamin B9 solution in 50 mM HEPES buffer into a sample cell containing 175 µL of 0.25 µM L. casei riboswitch solution in 50 mM HEPES buffer.
Figure 5: Zoomed-in version of Figure 1 excluding the first large negative slope before 300 seconds due to injection 1.
Analysis
The first injection took place at 0 seconds, and as shown in Figure 4, that injection caused a very large heat rate followed by what appears to be no further heat rate induced by the later injections. However, when this graph was zoomed in as shown in Figure 5, there were peaks with each of the following injections. This indicates that the first injection of Vitamin B9 interacted with most of the L. casei riboswitch present in the sample cell which left the following heat rate peaks to be very small in comparison because there was not enough sample for the titrant to have a comparable reaction with. Therefore, for the next experiment, the titrant concentration was reduced and tested.
Figure 6: ITC graph using 20 2.49 µL injections of 0.60 µM Vitamin B9 solution in 50 mM HEPES buffer into a sample cell containing 175 µL of 0.25 µM L. casei riboswitch solution in 50 mM HEPES buffer.
Although the first injection did not react with all of the sample as before and the first 19 samples showed large peaks, there was not a gradual decline of heat rate. Instead, the 20th injection peak had a much smaller magnitude which indicated that the sample ran out just then. This could be due to faulty stoichiometry predictions, non-ideal injection times, and/or still incorrect concentration ratios and will be looked into further.
